Methods for treating bone cancer pain by administering a nerve growth factor antagonist

ABSTRACT

The invention features methods and compositions for preventing or treating bone cancer pain including cancer pain associated with bone metastasis by administering an antagonist of nerve growth factor (NGF). The NGF antagonist may be an anti-NGF (such as anti-hNGF) antibody that is capable of binding hNGF.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of provisional patentapplications U.S. Ser. Nos. 60/620,654, filed Oct. 19, 2004, and60/560,781, filed Apr. 7, 2004, all of which are incorporated herein intheir entirety by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with U.S. Government support under NationalInstitutes of Health grants 5R37-NS23970-16, 5R01-DA11986-05 and1R01-NS048021-01A1. The U.S. Government may have certain rights in thisinvention.

FIELD OF THE INVENTION

The present invention relates to the use of a Nerve Growth Factor (NGF)antagonist for the prevention, amelioration, or treatment of bone cancerpain.

BACKGROUND OF THE INVENTION

Nerve growth factor (NGF) was the first neurotrophin to be identified,and its role in the development and survival of both peripheral andcentral neurons has been well characterized. NGF has been shown to be acritical survival and maintenance factor in the development ofperipheral sympathetic and embryonic sensory neurons and of basalforebrain cholinergic neurons (Smeyne, et al., Nature 368:246–249(1994); Crowley, et al., Cell 76:1001–1011 (1994)). NGF upregulatesexpression of neuropeptides in sensory neurons (Lindsay, et al., Nature337:362–364 (1989)), and its activity is mediated through two differentmembrane-bound receptors, the TrkA tyrosine kinase receptor and the p75receptor which is structurally related to other members of the tumornecrosis factor receptor family (Chao, et al., Science 232:518–521(1986)).

In addition to its effects in the nervous system, NGF has beenincreasingly implicated in processes outside of the nervous system. Forexample, NGF has been shown to enhance vascular permeability in the rat(Otten, et al., Eur J Pharmacol. 106:199–201 (1984)), enhance T- andB-cell immune responses (Otten, et al., Proc. Natl. Acad. Sci. USA86:10059–10063 (1989)), induce lymphocyte differentiation and mast cellproliferation and cause the release of soluble biological signals frommast cells (Matsuda, et al., Proc. Natl. Acad. Sci. USA 85:6508–6512(1988); Pearce, et al., J. Physiol. 372:379–393 (1986); Bischoff, etal., Blood 79:2662–2669 (1992); Horigome, et al., J. Biol. Chem.268:14881–14887(1993)). Although exogenously added NGF has been shown tobe capable of having all of these effects, it is important to note thatit has only rarely been shown that endogenous NGF is important in any ofthese processes in vivo (Torcia, et al., Cell. 85(3):345–56 (1996)).Therefore, it is not clear what the effect might be, if any, ofinhibiting the bioactivity of endogenous NGF.

NGF is produced by a number of cell types including mast cells (Leon, etal., Proc. Natl. Acad. Sci. USA 91:3739–3743 (1994)), B-lymphocytes(Torcia, et al., Cell 85:345–356 (1996), keratinocytes (Di Marco, etal., J. Biol. Chem. 268:22838–22846)), smooth muscle cells (Ueyama, etal., J. Hypertens. 11:1061–1065 (1993)), fibroblasts (Lindholm, et al.,Eur. J. Neurosci. 2:795–801 (1990)), bronchial epithelial cells (Kassel,et al., Clin, Exp. Allergy 31:1432–40 (2001)), renal mesangial cells(Steiner, et al., Am. J. Physiol. 261:F792–798 (1991)) and skeletalmuscle myotubes (Schwartz, et al., J Photochem, Photobiol. B 66:195–200(2002)). NGF receptors have been found on a variety of cell typesoutside of the nervous system. For example, TrkA has been found on humanmonocytes, T- and B-lymphocytes and mast cells.

An association between increased NGF levels and a variety ofinflammatory conditions has been observed in human patients as well asin several animal models. These include systemic lupus erythematosus(Bracci-Laudiero, et al., Neuroreport 4:563–565 (1993)), multiplesclerosis (Bracci-Laudiero, et al., Neurosci. Lett. 147:9–12 (1992)),psoriasis (Raychaudhuri, et al., Acta Derm. l'enereol. 78:84–86 (1998)),arthritis (Falcimi, et al., Ann. Rheum. Dis. 55:745–748 (1996)),interstitial cystitis (Okragly, et al., J. Urology 161:438–441 (1991)),asthma (Braun, et al., Eur. J Immunol. 28:3240–3251 (1998)),pancreatits, and prostatitis.

Consistently, an elevated level of NGF in peripheral tissues isassociated with inflammation and has been observed in a number of formsof arthritis. The synovium of patients affected by rheumatoid arthritisexpresses high levels of NGF while in non-inflamed synovium NGF has beenreported to be undetectable (Aloe, et al., Arch. Rheum. 35:351–355(1992)). Similar results were seen in rats with experimentally inducedrheumatoid arthritis (Aloe, et al., Clin. Exp. Rheumatol. 10:203–204(1992); Halliday et al., Neurochem. Res. 23:919–22 (1998)). Elevatedlevels of NGF have been reported in transgenic arthritic mice along withan increase in the number of mast cells. (Aloe, et al., Int. J. TissueReactions-Exp. Clin. Aspects 15:139–143 (1993)).

Treatment with exogenous NGF leads to an increase in pain and painsensitivity. This is illustrated by the fact that injection of NGF leadsto a significant increase in pain and pain sensitivity in both animalmodels (Lewin et al., J. Neurosci. 13:2136–2148 (1993); Amann, et al.,Pain 64, 323–329 (1996); Andreev, et al., Pain 63, 109–115 (1995)) andhuman (Dyck, et al., Neurology 48, 501–505 (1997); Petty, et al., AnnalsNeurol. 36, 244–246 (1994)). NGF appears to act by multiple mechanismsincluding inducing the neurotrophin BDNF (Apfel, et al., Mol. Cell.Neurosci. 7(2), 134–142 (1996); Michael, et al., J. Neurosci 17,8476–8490 (1997)) which in turn changes pain signal processing in thespinal cord (Hains, et al., Neurosci Lett. 320(3), 125–8 (2002);Miletic, et al., Neurosci Lett. 319(3), 137–40 (2002); Thompson, et al.,Proc Natl Acad Sci USA 96(14), 7714–8 (1999)), inducing changes in theperipheral and central connections of the sensory neurons and otherpain-transmitting neurons in the spinal cord (Lewin, et al., EuropeanJournal of Neuroscience 6, 1903–1912 (1994); Thompson, et al., Pain 62,219–231 (1995)), inducing changes in axonal growth (Lindsay, R M, JNeurosci. 8(7), 2394–405 (1988)) inducing bradykinin receptor expression(Peterson et al., Neuroscience 83:161–168 (1998)), inducing changes inexpression of genes responsible for nerve activation and conduction suchas ion channels (Boettger, et al., Brain 125(Pt 2), 252–63 (2002); Kerr,et al., Neuroreport 12(14), 3077–8 (2001); Gould, et al., Brain Res854(1–2), 19–29 (2000); Fjell et al., J. Neurophysiol. 81:803–810(1999)), potentiating the pain related receptor TRPV1 (Chuang, et al.,Nature 411 (6840), 957–62 (2001); Shu and Mendell, Neurosci. Lett.274:159–162 (1999)) and causing pathological changes in muscles (Foster,et al., J Pathol 197(2), 245–55 (2002)). Many of these changes takeplace directly on the pain transmitting sensory neurons and apparentlyare not dependent on concomitant inflammation. In addition, there are atleast two other cell types known to respond to NGF and that may beinvolved in changes of pain sensation or sensitivity. The first ofthese, the mast cell, has been reported to respond to NGF withdegranulation (Yan, et al., Clin. Sci. (Lond) 80:565–569 (1991)) or, inother studies, to cause or increase mediator production or release incollaboration with other agents (Pearce and Thompson, J. Physiol.372:379–393 (1986), Kawamoto, et al., J. Immunol. 168:6412–6419 (2002)).It has clearly been shown in the rat that NGF mediated pain responsesare at least somewhat mediated by mast cells (Lewin, et al., Eur. J.Neurosci. 6:1903–1912 (1994), Woolf, et al., J. Neurosci. 16:2716–2723(1996) although the potential relevance of this remains to be shown inhumans. Primary sympathetic neurons are also known to respond to NGF andto also be involved in pain signaling (Aley, et al., Neuroscience71:1083–1090 (1996)). It is clear that removing sympathetic innervationmodifies the hyperalgesia normally seen in response to treatment withNGF (Woolf, et al., J. Neurosci. 16:2716–2723 (1996)).

The use of NGF antagonists, such as anti-NGF antibody, to treat varioustypes of pain, has been described. See, e.g., U.S. Ser. Nos. 10/682,331,10/682,638, 10/682,332 (Pub. No. 2004/0131615), Ser. No. 10/783,730(Pub. No. 2004/0253244), Ser. No. 10/745,775 (Pub. No. 2004/0237124),Ser. No. 10/791,162; PCT/US03/32089 (WO 04/032870); PCT/US03/32083 (WO2005/000194); PCT/US03/32113; PCT/US2004/05162 (WO 04/073653);PCT/US03/41252 (WO 04/058184).

Bone cancer pain may arise in humans from either primary bone tumors ormore commonly from bone metastases (such as from breast, prostate, andlung carcinomas). See Luger et al., Pain 99:397–406 (2002). A mousemodel of bone cancer pain has been developed, and this model of bonecancer pain mirrors the pain observed in humans with moderate toadvanced bone cancer pain. See Luger et al., Pain 99:397–406 (2002);Clohisy et al., Clinical Orthopaedics and Related Research415S:S279–S288 (2003); Schwei et al., J. Neruosci. 19:10886–10897(1999); Honore et al., Nat. Med. 6: 521–529 (2000). Papers by Honore etal. and Schwei et al. state that the neurochemical signature of observedchanges in the spinal cord and DRG of bone cancer bearing animals isunique and distinguishable from either typical inflammatory pain ortypical neuropathic pain although there seem to be components of thisbiochemical signature similar to classic inflammatory and neuropathicpain states in this model. Honore et al. Neuroscience 98:585–598 (2000);Schwei et al. J. Neruosci. 19:10886–10897 (1999); Luger et al., Pain99:397–406 (2002).

All references cited herein, including patent applications andpublications, are incorporated by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION

The present invention is based upon the discovery that antagonists ofNGF, such as an anti-NGF antibody, are effective in treating bone cancerpain including cancer pain associated with bone metastasis. Thetreatment addresses one or more aspects of bone cancer pain includingcancer pain associated with bone metastasis as described herein.

In one aspect, the invention features a method for preventing ortreating bone cancer pain including cancer pain associated with bonemetastasis (also termed “bone metastasis pain”) by administering anantagonist of nerve growth factor (NGF). In some embodiments, the NGFantagonist is co-administered with an opioid analgesic. In someembodiments, the NGF antagonist is co-administered with an NSAID. Insome embodiments, the NGF antagonist is co-administered with an opioidanalgesic and an NSAID. In some embodiments, the NGF antagonist is notco-administered with an opioid analgesic. In some embodiments, the NGFantagonist is not co-administered with an NSAID.

In another aspect, the invention provides methods for reducing incidenceof bone cancer pain including cancer pain associated with bonemetastasis, ameliorating bone cancer pain including cancer painassociated with bone metastasis, palliating bone cancer pain includingcancer pain associated with bone metastasis; and/or delaying thedevelopment or progression of bone cancer pain including cancer painassociated with bone metastasis in an individual, said methodscomprising administering an effective amount of an NGF antagonist. Insome embodiments, the NGF antagonist is co-administered with an opioidanalgesic. In some embodiments, the NGF antagonist is co-administeredwith an NSAID. In some embodiments, the NGF antagonist isco-administered with an opioid analgesic and an NSAID. In someembodiments, the NGF antagonist is not co-administered with an opioidanalgesic. In some embodiments, the NGF antagonist is notco-administered with an NSAID.

In some embodiments, the bone cancer pain is from cancer originated inbone. In some embodiments, the bone cancer pain is from osteosarcoma. Insome embodiments, the bone cancer pain is from cancer metastasized tobone. In some embodiments, the bone metastasis is prostate cancermetastasized to bone. In some embodiments, the bone metastasis is breastcancer metastasized to bone. In some embodiments, the bone metastasis islung cancer metastasized to bone. In some embodiments, the bonemetastasis is sarcoma metastasized to bone. In some embodiments, thebone metastasis is kidney cancer metastasized to bone. In someembodiments, the bone metastasis is multiple myeloma metastasized tobone. In some embodiments, the cancer pain treated is mild to moderate.In some embodiments, the cancer pain treated is moderate to severe. Insome embodiments, the cancer pain treated is severe.

An NGF antagonist suitable for use in the methods of the invention isany agent that can directly or indirectly result in decreased NGFbiological activity. In some embodiments, an NGF antagonist (e.g., anantibody) binds (physically interacts with) NGF, binds to an NGFreceptor (such as trkA receptor and/or p75), and/or reduces (impedesand/or blocks) downstream NGF receptor signaling (e.g., inhibitors ofkinase signaling). Accordingly, in some embodiments, an NGF antagonistbinds (physically interacts with) NGF. In other embodiment, an NGFantagonist binds to an NGF receptor (such as TrkA receptor and/or p75).In other embodiments, an NGF antagonist reduces (impedes and/or blocks)downstream NGF receptor signaling (e.g., inhibitors of kinasesignaling). In other embodiments, an NGF antagonist inhibits (reduces)NGF synthesis and/or release. In another embodiment, the NGF antagonistis a TrkA immunoadhesin. In some embodiments, the NGF antagonist bindsNGF (such as hNGF) and does not significantly bind to relatedneurotrophins, such as NT-3, NT4/5, and/or BDNF. In some embodiments,the NGF antagonist is selected from any one or more of the following: ananti-NGF antibody, an anti-sense molecule directed to an NGF (includingan anti-sense molecule directed to a nucleic acid encoding NGF), ananti-sense molecule directed toward an NGF receptor (such as trkA and/orp75) (including an anti-sense molecule directed to a nucleic acidencoding an NGF receptor), an NGF inhibitory compound, an NGF structuralanalog, a dominant-negative mutation of a TrkA and/or p75 receptor thatbinds an NGF, an anti-TrkA antibody, an anti-p75 antibody, and a kinaseinhibitor. In another embodiment, the NGF antagonist is an anti-NGFantibody. In still other embodiments, the anti-NGF antibody is humanized(such as antibody E3 described herein). In some embodiments, theanti-NGF antibody is antibody E3 (as described herein). In otherembodiments, the anti-NGF antibody comprises one or more CDR(s) ofantibody E3 (such as one, two, three, four, five, or, in someembodiments, all six CDRs from E3). In other embodiments, the antibodyis human. In some embodiments, the antibody comprises three CDRs fromthe heavy chain of E3. In some embodiments, the antibody comprises threeCDRs from the light chain of E3. In still other embodiments, theanti-NGF antibody comprises the amino acid sequence of the heavy chainvariable region shown in Table 1 (SEQ ID NO:1). In still otherembodiments, the anti-NGF antibody comprises the amino acid sequence ofthe light chain variable region shown in Table 2 (SEQ ID NO:2) In stillother embodiments, the anti-NGF antibody comprises the amino acidsequence of the heavy chain variable region shown in Table 1 (SEQ IDNO:1) and the amino acid sequence of the light chain variable regionshown in Table 2 (SEQ ID NO:2). In still other embodiments, the antibodycomprises a modified constant region, such as a constant region that isimmunologically inert, e.g., does not trigger complement mediated lysis,or does not stimulate antibody-dependent cell mediated cytotoxicity(ADCC). In other embodiments, the constant region is modified asdescribed in Eur. J. Immunol. (1999) 29:2613–2624; PCT Application No.PCT/GB99/01441; and/or UK Patent Application No. 9809951.8.

In some embodiments, the NGF antagonist binds to NGF. In still otherembodiments, the NGF antagonist is an antibody that binds specificallyto NGF (such as human NGF). In still other embodiments, the antibodybinds essentially the same NGF epitope 6 as an antibody selected fromany one or more of the following mouse monoclonal antibodies: Mab 911,MAb 912 and MAb 938 (See Hongo, et al., Hybridoma 19:215–227 (2000)). Insome embodiments, the NGF antagonist binds to the trkA receptor. The NGFantagonist may be an anti-human NGF (anti-hNGF) monoclonal antibody thatis capable of binding hNGF and effectively inhibiting the binding ofhNGF to human TrkA (hTrkA) and/or effectively inhibiting activation ofhuman TrkA receptor.

The binding affinity of an anti-NGF antibody to NGF (such as hNGF) canbe about 0.10 to about 1.0 nM, about 0.10 nM to about 0.80 nM, about0.15 to about 0.75 nM and about 0.18 to about 0.72 nM. In oneembodiment, the binding affinity is between about 2 pM and 22 pM. Insome embodiment, the binding affinity is about 10 nM. In otherembodiments, the binding affinity is less than about 10 nM. In otherembodiments, the binding affinity is about 0.1 nM or about 0.07 nM. Inother embodiments, the binding affinity is less than about 0.1 nM, orless than about 0.07 nM. In other embodiments, the binding affinity isany of about 100 nM, about 50 nM, about 10 nM, about 1 nM, about 500 pM,about 100 pM, or about 50 pM to any of about 2 pM, about 5 pM, about 10pM, about 15 pM, about 20 pM, or about 40 pM. In some embodiments, thebinding affinity is any of about 100 nM, about 50 nM, about 10 nM, about1 nM, about 500 pM, about 100 pM, or about 50 pM, or less than about 50pM. In some embodiments, the binding affinity is less than any of about100 nM, about 50 nM, about 10 nM, about 1 nM, about 500 pM, about 100pM, or about 50 pM. In still other embodiments, the binding affinity isabout 2 pM, about 5 pM, about 10 pM, about 15 pM, about 20 pM, about 40pM, or greater than about 40 pM. As is well known in the art, bindingaffinity can be expressed as K_(D), or dissociation constant, and anincreased binding affinity corresponds to a decreased K_(D). The bindingaffinity of anti-NGF mouse monoclonal antibody 911 (Hongo et al.,Hybridoma 19:215–227 (2000) to human NGF is about 10 nM, and the bindingaffinity of humanized anti-NGF antibody E3 (described herein) to humanNGF is about 0.07 nM. Binding affinities for antibody 911 and E3 weremeasured using their Fab fragments.

The NGF antagonist may be administered prior to, during, and/or after anindividual has been diagnosed with bone cancer or cancer hasmetastasized to bone. Administration of an NGF antagonist can be by anymeans known in the art, including: orally, intravenously,subcutaneously, intraarterially, intramuscularly, intracardially,intraspinally, intrathoracically, intraperitoneally, intraventricularly,sublingually, and/or transdermally. In some embodiments, the NGFantagonist is an anti-NGF antibody, and administration is by one or moreof the following means: intravenously, subcutaneously, via inhalation,intraarterially, intramuscularly, intracardially, intraventricularly,and intraperitoneally. Administration may be systemic, e.g.intravenously, or localized.

In some embodiments, the NGF antagonist is administered in a dose ofabout 0.1 to 10 mg/kg of body weight, and in other embodiments, the NGFantagonist is administered in a dose of about 0.3 to 2.0 mg/kg of bodyweight.

In another aspect, the invention features a composition for treatingand/or preventing bone cancer pain including cancer pain associated withbone metastasis comprising an effective amount of a nerve growth factor(NGF) antagonist, in combination with one or more pharmaceuticallyacceptable excipients. In some embodiments, the NGF antagonist isco-administered with an opioid analgesic. In some embodiments, the NGFantagonist is co-administered with an NSAID. In some embodiments, theNGF antagonist is not co-administered with an opioid analgesic or anNSAID. In some embodiments, the NGF antagonist is an antibody thatspecifically binds to the NGF molecule. In other embodiments, the NGFantagonist is any antagonist described herein.

In another aspect, the invention features a kit for use in any of themethods described herein. In some embodiments, the kit comprises any ofthe NGF antagonists described herein, in combination with apharmaceutically acceptable carrier. In other embodiments, the kitfurther comprises instructions for use of the NGF antagonist in any ofthe methods described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting ongoing pain as assessed by spontaneousguarding and spontaneous flinching during a 2-min observation period onday 10 and day 14 post-sarcoma injection. “Naive” refers to animalswithout any injection. “Sham+veh.” refers to animals injected withα-minimum essential media into the femur marrow cavity and laterinjected with saline. “Sarc+veh.” refers to animals injected withsarcoma into the femur marrow cavity and later injected with saline.“Sarc+Anti-NGF” refers to animals injected with sarcoma into the femurmarrow cavity and later injected with anti-NGF antibody 911.

FIG. 2 is a graph depicting ambulatory pain as assessed by limb use andforced ambulatory guarding (rotarod) on day 10 and day 14 post-sarcomainjection. “Naive” refers to animals without any injection. “Sham+veh.”refers to animals injected with α-minimum essential media into the femurmarrow cavity and later injected with saline. “Sarc+veh.” refers toanimals injected with sarcoma into the femur marrow cavity and laterinjected with saline. “Sarc+Anti-NGF” refers to animals injected withsarcoma into the femur marrow cavity and later injected with anti-NGFantibody 911.

FIG. 3 is a graph depicting touch-evoked pain as assessed bypalpation-induced guarding and palpation-induced flinching during a2-min observation period on day 10 and day 14 post-sarcoma injection.“Naive” refers to animals without any injection. “Sham+veh.” refers toanimals injected with α-minimum essential media into the femur marrowcavity and later injected with saline. “Sarc+veh.” refers to animalsinjected with sarcoma into the femur marrow cavity and later injectedwith saline. “Sarc+Anti-NGF” refers to animals injected with sarcomainto the femur marrow cavity and later injected with anti-NGF antibody911.

FIG. 4 shows photographs demonstrating that the anti-NGF antibody had noeffect on disease progression in bone at day 14 (d14) post tumorinjection. Sham animals (n=8), given vehicle (sham+vehicle), are shownin (a) and (d); sarcoma (GFP transfected) injected animals (n=13), givenvehicle (sarcoma+vehicle) are shown in (b) and (e); and sarcoma (GFPtransfected) injected animals (n=8), given the anti-NGF antibody(sarcoma+anti-NGF), are shown in (c) and (f). FIGS. 4 a, 4 b, and 4 care radiographs showing presence or absence of bone destruction. FIGS. 4d, 4 e, and 4 f are photographs showing immunostaining with anti-GFPantibody. Scale bars: 1 mm.

FIG. 5 shows photographs demonstrating that the anti-NGF antibodytreatment had no observable effect on sensory innervation in skin.Hindpaw skin samples of both sarcoma-injected (a, b) and naïve (c, d)mice were immunostained for the neuropeptide calcitonin gene-relatedpeptide (CGRP), which labels unmyelinated peptidergic sensory nervefibers. Immunostaining of CGRP of hindpaw skin samples from sarcomainjected and vehicle treated (a, n=3) mice, sarcoma injected andanti-NGF antibody treated (b, n=8) mice, naïve and vehicle treated (c,n=8) mice, and naïve and anti-NGF antibody treated (d, n=8) mice areshown. Scale bar: 50 μm.

FIG. 6 shows graphs demonstrating that anti-NGF treatment attenuatedbone cancer pain. The time spent guarding and number of spontaneousflinches of the sarcoma injected limb over a 2-minute observation periodwas used as a measure of ongoing pain 8, 10, 12 and 14 days afterinjection and confinement of sarcoma cells to the left femur (a, b).Parameters of movement-evoked pain included quantification of time spentguarding and the number of flinches over a 2-minute observation periodfollowing a normally non-noxious palpation of the sarcoma-injected femur(c, d). “#” indicates P<0.05 vs. sham+vehicle; and “*” indicates P<0.05vs. sarcoma+vehicle.

FIG. 7 shows graphs demonstrating that anti-NGF treatment had no effecton baseline thermal or mechanical thresholds and had greater efficacythan morphine (MS) in reducing bone cancer pain. FIGS. 7 a and 7 b showthermal sensitivity (a, n=8 for naïve+vehicle, n=8 for naïve+anti-NGF)measured by latency of paw withdrawal to a thermal stimulus andmechanical sensitivity (b, n=8 for naïve+vehicle, n=8 fornaïve+anti-NGF) measured by 50% threshold of mechanical stimulation ofanti-NGF treatment (10 mg/kg, i.p., every 5 days) in naïve mice. FIGS. 7c and 7 d show ongoing pain behaviors evaluated by measuring spontaneousguarding (c) over a 2-minute observation period, and movement-evokedpain assessed by measuring the time spent guarding (d) over a 2-minuteobservation period following normally non-noxious palpation of thedistal femur. Values of spontaneous guarding (c) and palpation-inducedguarding (d) for naïve, sham and vehicle treated, sarcoma injected andvehicle treated, sarcoma injected and morphine (n=8, 10 mg/kg i.p.administered 15 min prior to testing) treated, sarcoma injected andmorphine (n=8, 30 mg/kg i.p. administered 15 min prior to testing)treated, and sarcoma injected and anti-NGF antibody (n=8, 10 mg/kg,every 5 days, i.p. administered from 6 days to 14 days post-tumorinjection) treated mice are shown. Error bars represent S.E.M. “#”indicates P<0.05 vs. sham+vehicle (n=8); “*” indicates P<0.05 vs.sarcoma+vehicle; and “+” indicates P<0.05 vs. sarcoma+morphine.

FIG. 8 shows photographs demonstrating that treatment with anti-NGFantagonist antibody reduced neurochemical changes and macrophageinfiltration in dorsal root ganglia (DRG) of tumor-bearing animals.FIGS. 8 a and 8 b show immunofluorescent staining of activatingtranscription factor-3 (ATF-3) in the ipsilateral L2 DRG oftumor-bearing animals vehicle treated (a, n=8) and anti-NGF antibodytreated (b, n=8) fourteen days post tumor implantation. Bottom panelshows immunofluorescent staining of CD-68 indicating the density ofactivated and infiltrating macrophages around injured sensory neuronswithin the ipsilateral DRG of tumor-bearing animals vehicle treated (c,n=7) and anti-NGF antibody treated (d, n=7). Scale bars a–d=5 μm.

FIG. 9 shows micrographs demonstrating that neurochemical changesassociated with central sensitization were attenuated by administrationof anti-NGF. FIGS. 9A and 9B show immunostaining of dynorphin in thedorsal horn of the spinal cord of sarcoma injected and vehicle treatedmice (A, n=9) and sarcoma injected and anti-NGF antibody treated mice(B, n=4). FIGS. 9C and 9D show representative confocal images of c-Fosexpressing neurons of the spinal cord in sarcoma injected and vehicletreated mice (C, n=4) and sarcoma injected and anti-NGF antibody treatedmice (D, n=4) following a normally non-noxious palpation oftumor-bearing limbs. Scale bar: 150 μm for A and B; 200 μm for C and D.

FIG. 10 shows graphs demonstrating that anti-NGF therapy attenuatedprostate tumor-induced bone cancer pain. Anti-NGF treatment (10 mg/kg,i.p, given on days 7, 12, and 17 post tumor-injection) attenuatedongoing bone cancer pain behaviors beginning on day 7 post-tumorinjection throughout disease progression. The time spent guarding andnumber of spontaneous flinches in ACE-1 injected femurs over a 2-minuteobservation period were used as measures of ongoing pain (A, B).Anti-NGF (filled square) significantly reduced ongoing pain behaviors intumor-injected animals as compared to ACE-1+vehicle (open square), andwas reduced to close to sham levels at day 9 for all parameters(circle). Both guarding and flinching in the sham+vehicle animals weresignificantly different from ACE-1+vehicle animals across diseaseprogression. Anti-NGF treatment had no effect on basal thermal ormechanical responses as measured by latency of paw withdrawal to athermal stimulus or increase in threshold of mechanical stimulation (C,D). Anti-NGF treatment produced a greater reduction in ongoing painbehaviors at day 19 than 10 mg/kg or 30 mg/kg morphine (i.p., 15 minprior to testing) (E, F). Movement-evoked pain was measured byquantification of time spent guarding and the number of flinches over a2-minute observation period following a normally non-noxious palpationof the ACE-1-injected femur (G, H). Error bars represent S.E.M. For FIG.10A–F, “#” indicates P<0.05 vs. sham+vehicle; “*” indicates P<0.05 vs.ACE-1+vehicle; and “+” indicates P<0.05 vs. ACE-1+morphine. For FIGS.10G and 10H, “*” indicates P<0.01 vs. sham; and “#” indicates P<0.01 vs.ACE-1+vehicle.

FIG. 11 are photographs demonstrating that anti-NGF antibody treatmenthad no effect on tumor burden or tumor-induced bone remodeling. Shamanimals, given vehicle, (A) showed no radiographically or histologically(H&E) (D) apparent bone destruction at day 19, whereas ACE-1+vehicleanimals (B, E) and ACE-1+anti-NGF animals (C, F) showed significanttumor growth and bone remodeling when examined radiologically andhistologically. H=hematopoeitic cells; T=tumor; WB=ACE-1 induced boneformation; Scale bar=1.5 mm.

FIG. 12 are images demonstrating that anti-NGF therapy did notsignificantly reduce tumor-induced osteoclastogenesis. TRAP stainedimages of sham+vehicle (A), ACE-1+vehicle (B), and ACE-1+anti-NGF (C)illustrate that proliferation occurred in this model along regions oftumor-induced bone remodeling with an increase in the number ofosteoclasts per mm² of diaphyseal intramedullary area in both theanti-NGF and vehicle-treated animals as compared to sham+vehicle andnaïve+vehicle animals. There was no observable difference inhistological appearance of the osteoclasts along the tumor/boneinterface or macrophages throughout the tumor when anti-NGF-treatedanimals (C) were compared to vehicle treated animals (B). Sham+vehicle(A) animals presented osteoclast numbers and morphology, and macrophageswhich were not significantly different from naïve animals.Arrows=osteoclasts; Arrowheads=macrophages; MB=mineralized bone;H=hematopoeitic cells; T=tumor; Scale bar: 50 μm.

FIG. 13 are photographs demonstrating that anti-NGF therapy did notinfluence the density of calcitonin gene-related peptide immunoreactive(CGRP-IR) sensory fibers in the femur. There was no observabledifference in the levels of immunofluorescence or density of CGRP-IRfibers between ACE-1+vehicle (A) animals and ACE-1+anti-NGF (B) animals.Also note that there was maintenance of CGRP-IR fibers with anti-NGFtherapy. T=tumor; Scale bar: 50 μm.

FIG. 14 are photographs demonstrating that anti-NGF therapy did notinfluence the density of calcitonin gene-related peptide immunoreactive(CGRP-IR) sensory fibers in the hindpaw skin. There was no observabledifference in the levels of immunofluorescence or density of CGRP-IRfibers in skin between naïve+vehicle (A) mice and naïve+anti-NGF (B)mice exists. Similarly, there was no difference in the levels ofimmunofluorescence or density of CGRP-IR nerve fibers betweenACE-1+vehicle (C) animals and ACE-1+anti-NGF (D) animals. Also note thatthere was no difference in CGRP-IR nerve fibers between the naïve andACE-1 injected mice (A, B vs. C, D). Scale bar: 50 μm.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that in vivoadministration of a therapeutically effective amount of an NGFantagonist such as anti-NGF monoclonal antibody may be used to treatbone cancer pain including cancer pain associated with bone metastasis.The invention is based on observations in a mouse bone cancer model thatadministration of anti-NGF antagonist antibody is strikingly effectivein reducing both ongoing and movement-evoked bone cancer pain.

The invention features methods of preventing or treating bone cancerpain including cancer pain associated with bone metastasis in anindividual (both human and non-human) by administering an effectiveamount of an NGF antagonist such as an anti-NGF antibody, for instancean anti-human NGF (anti-hNGF) monoclonal antibody. In some embodiments,the NGF antagonist is co-administered with an opioid analgesic. In someembodiments, the NGF antagonist is co-administered with an NSAID. Insome embodiments, the NGF antagonist is not co-administered with anopioid analgesic. In some embodiments, the NGF antagonist is notco-administered with an NSAID.

In another aspect, the invention provides methods for ameliorating,delaying the development of and/or preventing the progression of bonecancer pain including cancer pain associated with bone metastasiscomprising administering an effective amount of an NGF antagonist to anindividual. In some embodiments, the NGF antagonist is co-administeredwith an opioid analgesic. In some embodiments, the NGF antagonist isco-administered with an NSAID. In some embodiments, the NGF antagonistis not co-administered with an opioid analgesic. In some embodiments,the NGF antagonist is not co-administered with an NSAID.

The invention also features compositions and kits for treating bonecancer pain including cancer pain associated with bone metastasiscomprising an NGF antagonist such as an anti-NGF antibody, for instancean anti-NGF monoclonal antibody, for use in any of the methods providedherein. In some embodiments, the anti-NGF antibody is capable ofeffectively inhibiting NGF binding to its TrkA and/or p75 receptor(s)and/or of effectively inhibiting NGF from activating its TrkA and/or p75receptor(s).

General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, Molecular Cloning: ALaboratory Manual, second edition (Sambrook, et al., 1989) Cold SpringHarbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methodsin Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook(J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I.Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P.Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture:Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell,eds., 1993–8) J. Wiley and Sons; Methods in Enzymology (Academic Press,Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C.Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.Miller and M. P. Calos, eds., 1987); Current Protocols in MolecularBiology (F. M. Ausubel, et al., eds., 1987); PCR: The Polymerase ChainReaction, (Mullis, et al., eds., 1994); Current Protocols in Immunology(J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology(Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers,1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D.Catty., ed., IRL Press, 1988–1989); Monoclonal antibodies: a practicalapproach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using antibodies: a laboratory manual (E. Harlow and D. Lane (ColdSpring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995).

Definitions

An “antibody” (interchangeably used in plural form) is an immunoglobulinmolecule capable of specific binding to a target, such as acarbohydrate, polynucleotide, lipid, polypeptide, etc., through at leastone antigen recognition site, located in the variable region of theimmunoglobulin molecule. As used herein, the term encompasses not onlyintact polyclonal or monoclonal antibodies, but also fragments thereof(such as Fab, Fab′, F(ab′)2, Fv), single chain (ScFv), mutants thereof,fusion proteins comprising an antibody portion, humanized antibodies,chimeric antibodies, diabodies, linear antibodies, single chainantibodies, multispecific antibodies (e.g., bispecific antibodies) andany other modified configuration of the immunoglobulin molecule thatcomprises an antigen recognition site of the required specificity. Anantibody includes an antibody of any class, such as IgG, IgA, or IgM (orsub-class thereof), and the antibody need not be of any particularclass. Depending on the antibody amino acid sequence of the constantdomain of its heavy chains, immunoglobulins can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. Theheavy-chain constant domains that correspond to the different classes ofimmunoglobulins are called alpha, delta, epsilon, gamma, and mu,respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.

A “monoclonal antibody” refers to a homogeneous antibody populationwherein the monoclonal antibody is comprised of amino acids (naturallyoccurring and non-naturally occurring) that are involved in theselective binding of an antigen. A population of monoclonal antibodiesis highly specific, being directed against a single antigenic site. Theterm “monoclonal antibody” encompasses not only intact monoclonalantibodies and full-length monoclonal antibodies, but also fragmentsthereof (such as Fab, Fab′, F(ab′)2, Fv), single chain (ScFv), mutantsthereof, fusion proteins comprising an antibody portion, humanizedmonoclonal antibodies, chimeric monoclonal antibodies, and any othermodified configuration of the immunoglobulin molecule that comprises anantigen recognition site of the required specificity and the ability tobind to an antigen. It is not intended to be limited as regards to thesource of the antibody or the manner in which it is made (e.g., byhybridoma, phage selection, recombinant expression, transgenic animals,etc.).

As used herein, the term “nerve growth factor” and “NGF” refers to nervegrowth factor and variants thereof that retain at least part of theactivity of NGF. As used herein, NGF includes all mammalian species ofnative sequence NGF, including human, canine, feline, equine, or bovine.

“NGF receptor” refers to a polypeptide that is bound by or activated byNGF. NGF receptors include the TrkA receptor and the p75 receptor of anymammalian species, including, but are not limited to, human, canine,feline, equine, primate, or bovine.

An “NGF antagonist” refers to any molecule that blocks, suppresses orreduces (including significantly) NGF biological activity, includingdownstream pathways mediated by NGF signaling, such as receptor bindingand/or elicitation of a cellular response to NGF. The term “antagonist”implies no specific mechanism of biological action whatsoever, and isdeemed to expressly include and encompass all possible pharmacological,physiological, and biochemical interactions with NGF whether direct orindirect, or whether interacting with NGF, its receptor, or throughanother mechanism, and its consequences which can be achieved by avariety of different, and chemically divergent, compositions. ExemplaryNGF antagonists include, but are not limited to, an anti-NGF antibody,an anti-sense molecule directed to an NGF (including an anti-sensemolecule directed to a nucleic acid encoding NGF), an NGF inhibitorycompound, an NGF structural analog, a dominant-negative mutation of aTrkA receptor that binds an NGF, a TrkA immunoadhesin, an anti-TrkAantibody, an anti-p75 antibody, and a kinase inhibitor. For purpose ofthe present invention, it will be explicitly understood that the term“antagonist” encompass all the previously identified terms, titles, andfunctional states and characteristics whereby the NGF itself, an NGFbiological activity (including but not limited to its ability to mediateany aspect of cancer pain associated with bone metastasis), or theconsequences of the biological activity, are substantially nullified,decreased, or neutralized in any meaningful degree. In some embodiments,an NGF antagonist (e.g., an antibody) binds (physically interact with)NGF, binds to an NGF receptor (such as trkA receptor and/or p75receptor), reduces (impedes and/or blocks) downstream NGF receptorsignaling, and/or inhibits (reduces) NGF synthesis, production orrelease. In other embodiments, an NGF antagonist binds NGF and preventsTrkA receptor dimerization and/or TrkA autophosphorylation. In otherembodiments, an NGF antagonist inhibits or reduces NGF synthesis and/orproduction (release). Examples of types of NGF antagonists are providedherein.

As used herein, an “anti-NGF antibody” refers to an antibody which isable to bind to NGF and inhibit NGF biological activity and/ordownstream pathway(s) mediated by NGF signaling.

A “TrkA immunoadhesin” refers to a soluble chimeric molecule comprisinga fragment of a TrkA receptor, for example, the extracellular domain ofa TrkA receptor and an immunoglobulin sequence, which retains thebinding specificity of the TrkA receptor.

“Biological activity” of NGF generally refers to the ability to bind NGFreceptors and/or activate NGF receptor signaling pathways. Withoutlimitation, a biological activity includes any one or more of thefollowing: the ability to bind an NGF receptor (such as p75 and/orTrkA); the ability to promote TrkA receptor dimerization and/orautophosphorylation; the ability to activate an NGF receptor signalingpathway; the ability to promote cell differentiation, proliferation,survival, growth, migration and other changes in cell physiology,including (in the case of neurons, including peripheral and centralneuron) change in neuronal morphology, synaptogenesis, synapticfunction, neurotransmitter and/or neuropeptide release and regenerationfollowing damage; and the ability to mediate cancer pain associated withbone metastasis.

As used herein, “treatment” is an approach for obtaining beneficial ordesired clinical results. For purposes of this invention, beneficial ordesired clinical results include, but are not limited to, one or more ofthe following: improvement in any aspect of the pain including lesseningseverity, alleviation of one or more symptoms associated with bonecancer pain (e.g., cancer pain associated with bone metastasis)including any aspect of bone cancer pain (such as shortening duration ofpain, and/or reduction of pain sensitivity or sensation).

An “effective amount” is an amount sufficient to effect beneficial ordesired clinical results including alleviation or reduction in pain. Forpurposes of this invention, an effective amount of an NGF antagonist isan amount sufficient to treat, ameliorate, reduce the intensity of orprevent bone cancer pain including cancer pain associated with bonemetastasis. In some embodiments, the “effective amount” may reduce thepain of ongoing pain and/or breakthrough pain (including ambulatory painand touch-evoked pain), and it may be administered before, during,and/or after cancer has metastasized to bone. In some embodiment, the“effective amount” is an amount sufficient to delay development of bonecancer pain including cancer pain associated with bone metastasis.

“Reducing incidence” of pain means any of reducing severity (which caninclude reducing need for and/or amount of (e.g., exposure to) otherdrugs and/or therapies generally used for these conditions), duration,and/or frequency (including, for example, delaying or increasing time tobone cancer pain including cancer pain associated with bone metastasisin an individual). As is understood by those skilled in the art,individuals may vary in terms of their response to treatment, and, assuch, for example, a “method of reducing incidence of bone cancer painincluding cancer pain associated with bone metastasis in an individual”reflects administering the NGF antagonist described herein based on areasonable expectation that such administration may likely cause such areduction in incidence in that particular individual.

“Ameliorating” bone cancer pain (such as cancer pain associated withbone metastasis) or one or more symptoms of bone cancer pain means alessening or improvement of one or more symptoms of a bone cancer painas compared to not administering an NGF antagonist. “Ameliorating” alsoincludes shortening or reduction in duration of a symptom.

“Palliating” bone cancer pain (such as cancer pain associated with bonemetastasis) or one or more symptoms of a bone cancer pain meanslessening the extent of one or more undesirable clinical manifestationsof bone cancer pain in an individual or population of individualstreated with an NGF antagonist in accordance with the invention.

As used therein, “delaying” the development of bone cancer painincluding cancer pain associated with bone metastasis means to defer,hinder, slow, retard, stabilize, and/or postpone progression of bonecancer pain including cancer pain associated with bone metastasis. Thisdelay can be of varying lengths of time, depending on the history of thedisease and/or individuals being treated. As is evident to one skilledin the art, a sufficient or significant delay can, in effect, encompassprevention, in that the individual does not develop bone cancer painincluding cancer pain associated with bone metastasis. A method that“delays” development of the symptom is a method that reduces probabilityof developing the symptom in a given time frame and/or reduces extent ofthe symptoms in a given time frame, when compared to not using themethod. Such comparisons are typically based on clinical studies, usinga number of subjects sufficient to give a statistically significantresult.

“Development” or “progression” of bone cancer pain including cancer painassociated with bone metastasis means initial manifestations and/orensuing progression of the disorder. Development of bone cancer painincluding cancer pain associated with bone metastasis can be detectableand assessed using standard clinical techniques as well known in theart. However, development also refers to progression that may beundetectable. For purpose of this invention, development or progressionrefers to the biological course of the symptoms. “Development” includesoccurrence, recurrence, and onset. As used herein “onset” or“occurrence” of bone cancer pain (such as cancer pain associated withbone metastasis) includes initial onset and/or recurrence.

As used herein, “co-administration” includes simultaneous administrationand/or administration at different times. Co-administration alsoencompasses administration as a co-formulation (i.e., the NGF antagonistand an agent are present in the same composition) or administration asseparate compositions. As used herein, co-administration is meant toencompass any circumstance wherein an agent and NGF antagonist areadministered to an individual, which can occur simultaneously and/orseparately. As further discussed herein, it is understood that the NGFantagonist and an agent can be administered at different dosingfrequencies or intervals. For example, an anti-NGF antibody can beadministered weekly, while the agent can be administered morefrequently. It is understood that the NGF antagonist and the agent canbe administered using the same route of administration or differentroutes of administration.

The term “opioid analgesic” refers to all drugs, natural or synthetic,with morphine-like actions. The synthetic and semi-synthetic opioidanalgesics are derivatives of five chemical classes of compound:phenanthrenes; phenylheptylamines; phenylpiperidines; morphinans; andbenzomorphans, all of which are within the scope of the term. Exemplaryopioid analgesics include codeine, dihydrocodeine, diacetylmorphine,hydrocodone, hydromorphone, levorphanol, oxymorphone, alfentanil,buprenorphine, butorphanol, fentanyl, sufentanyl, meperidine, methadone,nalbuphine, propoxyphene and pentazocine or pharmaceutically acceptablesalts thereof.

The term “NSAID” refers to a non-steroidal anti-inflammatory compound.NSAIDs are categorized by virtue of their ability to inhibitcyclooxygenase. Cyclooxygenase 1 and cyclooxygenase 2 are two majorisoforms of cyclooxygenase and most standard NSAIDs are mixed inhibitorsof the two isoforms. Most standard NSAIDs fall within one of thefollowing five structural categories: (1) propionic acid derivatives,such as ibuprofen, naproxen, naprosyn, diclofenac, and ketoprofen; (2)acetic acid derivatives, such as tolmetin and slindac; (3) fenamic acidderivatives, such as mefenamic acid and meclofenamic acid; (4)biphenylcarboxylic acid derivatives, such as diflunisal and flufenisal;and (5) oxicams, such as piroxim, sudoxicam, and isoxicam.

Another class of NSAID has been described which selectively inhibitcyclooxygenase 2. Cox-2 inhibitors have been described, e.g., in U.S.Pat. Nos. 5,616,601; 5,604,260; 5,593,994; 5,550,142; 5,536,752;5,521,213; 5,475,995; 5,639,780; 5,604,253; 5,552,422; 5,510,368;5,436,265; 5,409,944; and 5,130,311, all of which are herebyincorporated by reference. Certain exemplary COX-2 inhibitors includecelecoxib (SC-58635), DUP-697, flosulide (CGP-28238), meloxicam,6-methoxy-2 naphthylacetic acid (6-MNA), rofecoxib, MK-966, nabumetone(prodrug for 6-MNA), nimesulide, NS-398, SC-5766, SC-58215, T-614; orcombinations thereof.

An “individual” is a mammal, more preferably a human. Mammals include,but are not limited to, farm animals, sport animals, pets, primates,horses, dogs, cats, mice and rats.

Methods of the Invention

With respect to all methods described herein, reference to an NGFantagonist also includes compositions comprising one or more of theseagents. These compositions may further comprise suitable excipients,such as pharmaceutically acceptable excipients (carriers) includingbuffers, which are well known in the art. The present invention can beused alone or in combination with other conventional methods oftreatment.

Methods for Preventing or Treating Bone Cancer Pain Including CancerPain associated with Bone Metastasis

The present invention is useful for treating, delaying development ofand/or preventing bone cancer pain including cancer pain associated withbone metastasis in an individual, both human and non-human. The qualityof life in individuals having bone cancer may be improved.

Cancer metastasis to bone may be associated with a net bone formation ora net bone destruction. In some embodiments, the method of the inventionis used for treating bone cancer pain associated with a net boneformation (osteoblastic activity), such as for treating pain of prostatecancer metastasis to bone. In some embodiments, the method of theinvention is used for treating bone cancer pain associated with a netbone destruction (osteolytic activity), such as for treating pain ofsarcoma metastasis to bone.

Accordingly, in one aspect, the invention provides methods of treatingbone cancer pain including cancer pain associated with bone metastasisin an individual comprising administering an effective amount of an NGFantagonist, such as an anti-NGF antibody. In some embodiments, the NGFantagonist is co-administered with an opioid analgesic. In someembodiments, the NGF antagonist is co-administered with an NSAID. Insome embodiments, the NGF antagonist is co-administered with an opioidanalgesic and an NSAID. In some embodiments, the amount of the opioidanalgesic and/or the NSAID administered for pain alleviation arereduced, comparing to the amount administered in the absence of the NGFantagonist. Adverse effects due to the opioid analgesic and/or the NSAIDmay be reduced or eliminated when they are co-administered with the NGFantagonist. In some embodiments, the NGF antagonist is notco-administered with an opioid analgesic. In other embodiments, the NGFantagonist is not co-administered with an NSAID. In other embodiments,the NGF antagonist is not co-administered with an opioid analgesicand/or an NSAID.

In another aspect, the invention provides methods of preventing,ameliorating and/or preventing the development or progression of bonecancer pain including cancer pain associated with bone metastasis. Insome embodiments, the NGF antagonist is co-administered with an opioidanalgesic. In some embodiments, the NGF antagonist is co-administeredwith an NSAID. In some embodiments, the NGF antagonist isco-administered with an opioid analgesic and an NSAID. In someembodiments, the NGF antagonist is not co-administered with an opioidanalgesic. In other embodiments, the NGF antagonist is notco-administered with an NSAID. In other embodiments, the NGF antagonistis not co-administered with an opioid analgesic and/or an NSAID.

It is appreciated that although reference is generally made herein totreating or preventing bone cancer pain such as cancer pain associatedwith bone metastasis, the NGF antagonist can be administered before anevent or condition(s) with an increased risk of bone cancer pain.

An NGF antagonist may be administered in conjunction with othertherapies for bone cancer, such as radiation, and chemotherapy. The NGFantagonist may also be administered in conjunction with other analgesicsused for bone cancer pain. Examples of such analgesics arebisphosphonates (e.g., Alendronate), gabapentin, and radiation. Theamount of these analgesics administered for bone cancer pain alleviationmay be reduced, comparing to the amount administered in the absence ofthe NGF antagonist. Adverse effects due to these analgesics may bereduced or eliminated when they are co-administered with the NGFantagonist.

Diagnosis or assessment of pain is well-established in the art.Assessment may be performed based on objective measure, such asobservation of behavior such as reaction to stimuli, facial expressionsand the like. Assessment may also be based on subjective measures, suchas patient characterization of pain using various pain scales. See,e.g., Katz et al, Surg Clin North Am. (1999) 79 (2):231–52; Caraceni etal. J Pain Symptom Manage (2002) 23(3):239–55.

NGF Antagonists

The methods of the invention use an NGF antagonist, which refers to anymolecule that blocks, suppresses or reduces (including significantly)NGF biological activity, including downstream pathways mediated by NGFsignaling, such as receptor binding and/or elicitation of a cellularresponse to NGF. The term “antagonist” implies no specific mechanism ofbiological action whatsoever, and is deemed to expressly include andencompass all possible pharmacological, physiological, and biochemicalinteractions with NGF and its consequences which can be achieved by avariety of different, and chemically divergent, compositions. ExemplaryNGF antagonists include, but are not limited to, an anti-NGF antibody,an anti-sense molecule directed to NGF (including an anti-sense moleculedirected to a nucleic acid encoding NGF), an anti-sense moleculedirected to an NGF receptor (such as TrkA receptor and/or p75 receptor)(including an anti-sense molecule directed to a nucleic acid encodingTrkA and/or p75), an NGF inhibitory compound, an NGF structural analog,a dominant-negative mutation of a TrkA receptor that binds an NGF, aTrkA immunoadhesin, an anti-TrkA antibody, a dominant-negative mutationof a p75 receptor that binds an NGF, an anti-p75 antibody, and a kinaseinhibitor. For purpose of the present invention, it will be explicitlyunderstood that the term “antagonist” encompasses all the previouslyidentified terms, titles, and functional states and characteristicswhereby the NGF itself, an NGF biological activity (including but notlimited to its ability to mediate any aspect of cancer pain associatedwith bone metastasis), or the consequences of the biological activity,are substantially nullified, decreased, or neutralized in any meaningfuldegree. In some embodiments, an NGF antagonist (e.g., an antibody) binds(physically interact with) NGF, binds to an NGF receptor (such as TrkAreceptor and/or p75 receptor), and/or reduces (impedes and/or blocks)downstream NGF receptor signaling. Accordingly, in some embodiments, anNGF antagonist binds (physically interacts with) NGF. In someembodiments, the NGF antagonist is a polypeptide which binds to NGF. Insome embodiments, the NGF antagonist is a peptide or a modified peptide(such as NGF binding peptide fused to a Fc domain) described in PCT WO2004/026329. In other embodiment, an NGF antagonist binds to an NGFreceptor (such as trkA receptor or p75). In other embodiments, an NGFantagonist reduces (impedes and/or blocks) downstream NGF receptorsignaling (e.g., inhibitors of kinase signaling and inhibitors ofdownstream signaling cascade). In other embodiments, an NGF antagonistinhibits (reduces) NGF synthesis and/or release. In another embodiment,the NGF antagonist is an NGF antagonist that is not a TrkA immunoadhesin(i.e., is other than a TrkA immunoadhesin). In another embodiment, theNGF antagonist is other than an anti-NGF antibody. In other embodiment,the NGF antagonist is other than a TrkA immunoadhesin and other than ananti-NGF antibody. In some embodiment, the NGF antagonist binds NGF(such as hNGF) and does not significantly bind to related neurotrophins,such as NT-3, NT4/5, and/or BDNF. In some embodiments, the NGFantagonist is not associated with an adverse immune response. In otherembodiments, the NGF antagonist is an anti-NGF antibody. In still otherembodiments, the anti-NGF antibody is humanized (such as antibody E3described herein). In some embodiments, the anti-NGF antibody isantibody E3 (as described herein). In other embodiments, the anti-NGFantibody comprises one or more CDR(s) of antibody E3 (such as one, two,three, four, five, or, in some embodiments, all six CDRs from E3). Inother embodiments, the antibody is human. In some embodiments, theantibody is a human anti-NGF neutralizing antibody described in WO2005/019266. In still other embodiments, the anti-NGF antibody comprisesthe amino acid sequence of the heavy chain variable region shown inTable 1 (SEQ ID NO:1) and the amino acid sequence of the light chainvariable region shown in Table 2 (SEQ ID NO:2). In still otherembodiments, the antibody comprises a modified constant region, such asa constant region that is immunologically inert, e.g., does not triggercomplement mediated lysis, or does not stimulate antibody-dependent cellmediated cytotoxicity (ADCC). In other embodiments, the constant regionis modified as described in Eur. J. Immunol. (1999) 29:2613–2624; PCTApplication No. PCT/GB99/01441; and/or UK Patent Application No.9809951.8.

Anti-NGF Antibodies

In some embodiments of the invention, the NGF antagonist comprises ananti-NGF antibody. An anti-NGF antibody should exhibit any one or moreof the following characteristics: (a) bind to NGF and inhibit NGFbiological activity and/or downstream pathways mediated by NGF signalingfunction; (b) prevent, ameliorate, or treat any aspect of bone cancerpain including cancer pain associated with bone metastasis; (c) block ordecrease NGF receptor activation (including TrkA receptor dimerizationand/or autophosphorylation); (d) increase clearance of NGF; (e) inhibit(reduce) NGF synthesis, production or release.

Anti-NGF antibodies are known in the art, see, e.g., PCT PublicationNos. WO 01/78698, WO 01/64247, U.S. Pat. Nos. 5,844,092, 5,877,016, and6,153,189; Hongo et al., Hybridoma, 19:215–227 (2000); Cell. Molec.Biol. 13:559–568 (1993); GenBank Accession Nos. U39608, U39609, L17078,or L17077.

In some embodiments, the anti-NGF antibody is a humanized mouse anti-NGFmonoclonal antibody termed antibody “E3” (PCT WO 04/058184), whichcomprises the human heavy chain IgG2a constant region containing thefollowing mutations: A330P331 to S330S331 (amino acid numbering withreference to the wildtype IgG2a sequence; see Eur. J. Immunol. (1999)29:2613–2624); the human light chain kappa constant region; and theheavy and light chain variable regions shown in Tables 1 and 2.

TABLE 1 Heavy chain variable regionQVQLQESGPGLVKPSETLSLTCTVSGFSLIGYDLNWI (SEQ ID NO:1)RQPPGKGLEWIGIIWGDGTTDYNSAVKSRVTISKDTSKNQFSLKLSSVTAADTAVYYCARGGYWYATSYYFDYW GQGTLVTVS.

TABLE 2 Light chain variable regionDIQMTQSPSSLSASVGDRVTITCRASQSISNNLNWYQ (SEQ ID NO:2)QKPGKAPKLLIYYTSRFHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQEHTLPYTFGQGTKLEIKRT.

The following polynucleotides encoding the heavy chain variable regionor the light chain variable region were deposited at the ATCC on Jan. 8,2003:

Material ATCC Accession No. Date of Deposit Vector Eb.911.3E E3 lightchain V region PTA-4893 Jan. 8, 2003 Vector Eb.pur.911.3E E3 light chainV region PTA-4894 Jan. 8, 2003 Vector Db.911.3E E3 heavy chain VPTA-4895 Jan. 8, 2003 region

Vector Eb.911.3E is a polynucleotide encoding the light chain variableregion shown in Table 2; vector Eb.pur.911.3E is a polynucleotideencoding the light chain variable region shown in Table 2 and vectorDb.911.3E is a polynucleotide encoding the heavy chain variable regionshown in Table 1. These polynucleotides also encode constant domains.

There are at least two techniques for determining CDRs: (1) an approachbased on cross-species sequence variability (i.e., Kabat et al.Sequences of Proteins of Immunological Interest, (5th ed., 1991,National Institutes of Health, Bethesda Md.)); and (2) an approach basedon crystallographic studies of antigen-antibody complexes (Chothia etal. (1989) Nature 342:877; Al-lazikani et al (1997) J. Molec. Biol.273:927–948)). As used herein, a CDR may refer to CDRs defined by eitherapproach or by a combination of both approaches.

In another embodiment, the anti-NGF antibody comprises one or moreCDR(s) of antibody E3 (such as one, two, three, four, five, or, in someembodiments, all six CDRs from E3). Determination of CDR regions is wellwithin the skill of the art. CDR(s) may be Kabat, Chothia, or acombination of Kabat and Chothia.

The antibodies useful in the present invention can encompass monoclonalantibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab′,F(ab′)2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies,heteroconjugate antibodies, single chain (ScFv), mutants thereof, fusionproteins comprising an antibody portion, humanized antibodies, and anyother modified configuration of the immunoglobulin molecule thatcomprises an antigen recognition site of the required specificity,including glycosylation variants of antibodies, amino acid sequencevariants of antibodies, and covalently modified antibodies. Theantibodies may be murine, rat, human, or any other origin (includingchimeric or humanized antibodies). For purposes of this invention, theantibody reacts with NGF in a manner that inhibits NGF and/or downstreampathways mediated by the NGF signaling function. In one embodiment, theantibody is a human antibody which recognizes one or more epitopes onhuman NGF. In another embodiment, the antibody is a mouse or ratantibody which recognizes one or more epitopes on human NGF. In anotherembodiment, the antibody recognizes one or more epitopes on an NGFselected from the group consisting of: primate, canine, feline, equine,and bovine. In other embodiments, the antibody comprises a modifiedconstant region, such as a constant region that is immunologicallyinert, e.g., does not trigger complement mediated lysis, or does notstimulate antibody-dependent cell mediated cytotoxicity (ADCC). ADCCactivity can be assessed using methods disclosed in U.S. Pat. No.5,500,362. In other embodiments, the constant region is modified asdescribed in Eur. J. Immunol. (1999) 29:2613–2624; PCT Application No.PCT/GB99/01441; and/or UK Patent Application No. 9809951.8.

The binding affinity of an anti-NGF antibody to NGF (such as hNGF) canbe about 0.10 to about 0.80 nM, about 0.15 to about 0.75 nM and about0.18 to about 0.72 nM. In one embodiment, the binding affinity isbetween about 2 pM and 22 pM. In some embodiment, the binding affinityis about 10 nM. In other embodiments, the binding affinity is less thanabout 10 nM. In other embodiments, the binding affinity is about 0.1 nMor about 0.07 nM. In other embodiments, the binding affinity is lessthan about 0.1 nM, or less than about 0.07 nM. In other embodiments, thebinding affinity is any of about 100 nM, about 50 nM, about 10 nM, about1 nM, about 500 pM, about 100 pM, or about 50 pM to any of about 2 pM,about 5 pM, about 10 pM, about 15 pM, about 20 pM, or about 40 pM. Insome embodiments, the binding affinity is any of about 100 nM, about 50nM, about 10 nM, about 1 nM, about 500 pM, about 100 pM, or about 50 pM,or less than about 50 pM. In some embodiments, the binding affinity isless than any of about 100 nM, about 50 nM, about 10 nM, about 1 nM,about 500 pM, about 100 pM, or about 50 pM. In still other embodiments,the binding affinity is about 2 pM, about 5 pM, about 10 pM, about 15pM, about 20 pM, about 40 pM, or greater than about 40 pM.

One way of determining binding affinity of antibodies to NGF is bymeasuring binding affinity of monofunctional Fab fragments of theantibody. To obtain monofunctional Fab fragments, an antibody (forexample, IgG) can be cleaved with papain or expressed recombinantly. Theaffinity of an anti-NGF Fab fragment of an antibody can be determined bysurface plasmon resonance (BIAcore3000™ surface plasmon resonance (SPR)system, BIAcore, INC, Piscaway N.J.). CM5 chips can be activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiinide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Human NGF (or any other NGF) can be diluted into 10 mM sodium acetate pH4.0 and injected over the activated chip at a concentration of 0.005mg/mL. Using variable flow time across the individual chip channels, tworanges of antigen density can be achieved: 100–200 response units (RU)for detailed kinetic studies and 500–600 RU for screening assays. Thechip can be blocked with ethanolamine. Regeneration studies have shownthat a mixture of Pierce elution buffer (Product No. 21004, PierceBiotechnology, Rockford Ill.) and 4 M NaCl (2:1) effectively removes thebound Fab while keeping the activity of hNGF on the chip for over 200injections. HBS-EP buffer (0.01M HEPES, pH 7.4, 0.15 NaCl, 3 mM EDTA,0.005% Surfactant P20) is used as running buffer for the BIAcore assays.Serial dilutions (0.1–10× estimated K_(D)) of purified Fab samples areinjected for 1 min at 100 μL/min and dissociation times of up to 2 h areallowed. The concentrations of the Fab proteins are determined by ELISAand/or SDS-PAGE electrophoresis using a Fab of known concentration (asdetermined by amino acid analysis) as a standard. Kinetic associationrates (k_(on)) and dissociation rates (k_(off)) are obtainedsimultaneously by fitting the data to a 1:1 Langmuir binding model(Karlsson, R. Roos, H. Fagerstam, L. Petersson, B. (1994). MethodsEnzymology 6. 99–110) using the BIAevaluation program. Equilibriumdissociation constant (K_(D)) values are calculated as k_(off)/k_(on).This protocol is suitable for use in determining binding affinity of anantibody to any NGF, including human NGF, NGF of another vertebrate (insome embodiments, mammalian) (such as mouse NGF, rat NGF, primate NGF),as well as for use with other neurotrophins, such as the relatedneurotrophins NT3, NT4/5, and/or BDNF.

In some embodiments, the antibody binds human NGF, and does notsignificantly bind an NGF from another vertebrate species (in someembodiments, mammalian). In some embodiments, the antibody binds humanNGF as well as one or more NGF from another vertebrate species (in someembodiments, mammalian). In still other embodiments, the antibody bindsNGF and does not significantly cross-react with other neurotrophins(such as the related neurotrophins, NT3, NT4/5, and/or BDNF). In someembodiments, the antibody binds NGF as well as at least one otherneurotrophin. In some embodiments, the antibody binds to a mammalianspecies of NGF, such as horse or dog, but does not significantly bind toNGF from anther mammalian species.

The epitope(s) can be continuous or discontinuous. In one embodiment,the antibody binds essentially the same hNGF epitopes as an antibodyselected from the group consisting of MAb 911, MAb 912, and MAb 938 asdescribed in Hongo et al., Hybridoma, 19:215–227 (2000). In anotherembodiment, the antibody binds essentially the same hNGF epitope as MAb911. In still another embodiment, the antibody binds essentially thesame epitope as MAb 909. Hongo et al., supra. For example, the epitopemay comprise one or more of: residues K32, K34 and E35 within variableregion 1 (amino acids 23–35) of hNGF; residues F79 and T81 withinvariable region 4 (amino acids 81–88) of hNGF; residues H84 and K88within variable region 4; residue R103 between variable region 5 (aminoacids 94–98) of hNGF and the C-terminus (amino acids 111–118) of hNGF;residue E11 within pre-variable region 1 (amino acids 10–23) of hNGF;Y52 between variable region 2 (amino acids 40–49) of hNGF and variableregion 3 (amino acids 59–66) of hNGF; residues L112 and S113 within theC-terminus of hNGF; residues R59 and R69 within variable region 3 ofhNGF; or residues V18, V20, and G23 within pre-variable region 1 ofhNGF. In addition, an epitope can comprise one or more of the variableregion 1, variable region 3, variable region 4, variable region 5, theN-terminus region, and /or the C-terminus of hNGF. In still anotherembodiment, the antibody significantly reduces the solvent accessibilityof residue R103 of hNGF. It is understood that although the epitopesdescribed above relate to human NGF, one of ordinary skill can align thestructures of human NGF with the NGF of other species and identifylikely counterparts to these epitopes.

In one aspect, antibodies (e.g., human, humanized, mouse, chimeric) thatcan inhibit NGF may be made by using immunogens that express full lengthor partial sequence of NGF. In another aspect, an immunogen comprising acell that overexpresses NGF may be used. Another example of an immunogenthat can be used is NGF protein that contains full-length NGF or aportion of the NGF protein.

The anti-NGF antibodies may be made by any method known in the art. Theroute and schedule of immunization of the host animal are generally inkeeping with established and conventional techniques for antibodystimulation and production, as further described herein. Generaltechniques for production of human and mouse antibodies are known in theart and are described herein.

It is contemplated that any mammalian subject including humans orantibody producing cells therefrom can be manipulated to serve as thebasis for production of mammalian, including human, hybridoma celllines. Typically, the host animal is inoculated intraperitoneally,intramuscularly, orally, subcutaneously, intraplantar, and/orintradermally with an amount of immunogen, including as describedherein.

Hybridomas can be prepared from the lymphocytes and immortalized myelomacells using the general somatic cell hybridization technique of Kohler,B. and Milstein, C. (1975) Nature 256:495–497 or as modified by Buck, D.W., et al., In Vitro, 18:377–381 (1982). Available myeloma lines,including but not limited to X63-Ag8.653 and those from the SalkInstitute, Cell Distribution Center, San Diego, Calif., USA, may be usedin the hybridization. Generally, the technique involves fusing myelomacells and lymphoid cells using a fusogen such as polyethylene glycol, orby electrical means well known to those skilled in the art. After thefusion, the cells are separated from the fusion medium and grown in aselective growth medium, such as hypoxanthine-aminopterin-thymidine(HAT) medium, to eliminate unhybridized parent cells. Any of the mediadescribed herein, supplemented with or without serum, can be used forculturing hybridomas that secrete monoclonal antibodies. As anotheralternative to the cell fusion technique, EBV immortalized B cells maybe used to produce the anti-NGF monoclonal antibodies of the subjectinvention. The hybridomas are expanded and subcloned, if desired, andsupernatants are assayed for anti-immunogen activity by conventionalimmunoassay procedures (e.g., radioimmunoassay, enzyme immunoassay, orfluorescence immunoassay).

Hybridomas that may be used as source of antibodies encompass allderivatives, progeny cells of the parent hybridomas that producemonoclonal antibodies specific for NGF, or a portion thereof.

Hybridomas that produce such antibodies may be grown in vitro or in vivousing known procedures. The monoclonal antibodies may be isolated fromthe culture media or body fluids, by conventional immunoglobulinpurification procedures such as ammonium sulfate precipitation, gelelectrophoresis, dialysis, chromatography, and ultrafiltration, ifdesired. Undesired activity if present, can be removed, for example, byrunning the preparation over adsorbents made of the immunogen attachedto a solid phase and eluting or releasing the desired antibodies off theimmunogen. Immunization of a host animal with a human NGF, or a fragmentcontaining the target amino acid sequence conjugated to a protein thatis immunogenic in the species to be immunized, e.g., keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsininhibitor using a bifunctional or derivatizing agent, for examplemaleimidobenzoyl sulfosuccinimide ester (conjugation through cysteineresidues), N-hydroxysuccinimide (through lysine residues),glytaradehyde, succinic anhydride, SOCl2, or R1N═C═NR, where R and R1are different alkyl groups, can yield a population of antibodies (e.g.,monoclonal antibodies).

If desired, the anti-NGF antibody (monoclonal or polyclonal) of interestmay be sequenced and the polynucleotide sequence may then be cloned intoa vector for expression or propagation. The sequence encoding theantibody of interest may be maintained in vector in a host cell and thehost cell can then be expanded and frozen for future use. In analternative, the polynucleotide sequence may be used for geneticmanipulation to “humanize” the antibody or to improve the affinity, orother characteristics of the antibody. For example, the constant regionmay be engineered to more resemble human constant regions to avoidimmune response if the antibody is used in clinical trials andtreatments in humans. It may be desirable to genetically manipulate theantibody sequence to obtain greater affinity to NGF and greater efficacyin inhibiting NGF. It will be apparent to one of skill in the art thatone or more polynucleotide changes can be made to the anti-NGF antibodyand still maintain its binding ability to NGF.

“Humanized” antibodies generally refer to a molecule having an antigenbinding site that is substantially derived from an immunoglobulin from anon-human species and the remaining immunoglobulin structure of themolecule based upon the structure and/or sequence of a humanimmunoglobulin. The antigen binding site may comprise either completevariable domains fused onto constant domains or only the complementaritydetermining regions (CDRs) grafted onto appropriate framework regions inthe variable domains. Antigen binding sites may be wild type or modifiedby one or more amino acid substitutions, e.g., modified to resemblehuman immunoglobulin more closely. Some forms of humanized antibodiespreserve all CDR sequences (for example, a humanized mouse antibodywhich contains all six CDRs from the mouse antibodies). Other forms ofhumanized antibodies have one or more CDRs (one, two, three, four, five,six) which are altered with respect to the original antibody. In someinstances, framework region (FR) residues or other residues of the humanimmunoglobulin replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are notfound in the recipient antibody or in the donor antibody.

There are four general steps to humanize a monoclonal antibody. Theseare: (1) determining the nucleotide and predicted amino acid sequence ofthe starting antibody light and heavy variable domains (2) designing thehumanized antibody, i.e., deciding which antibody framework region touse during the humanizing process (3) the actual humanizingmethodologies/techniques and (4) the transfection and expression of thehumanized antibody. See, for example, U.S. Pat. Nos. 4,816,567;5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761; 5,693,762;5,585,089; 6,180,370; and 6,548,640.

A number of “humanized” antibody molecules comprising an antigen-bindingsite derived from a non-human immunoglobulin have been described,including chimeric antibodies having rodent or modified rodent V regionsand their associated complementarity determining regions (CDRs) fused tohuman constant domains. See, for example, Winter et al. Nature349:293–299 (1991), Lobuglio et al. Proc. Nat. Acad. Sci. USA86:4220–4224 (1989), Shaw et al. J Immunol. 138:4534–4538 (1987), andBrown et al. Cancer Res. 47:3577–3583 (1987). Other references describerodent CDRs grafted into a human supporting framework region (FR) priorto fusion with an appropriate human antibody constant domain. See, forexample, Riechmann et al. Nature 332:323–327 (1988), Verhoeyen et al.Science 239:1534–1536 (1988), and Jones et al. Nature 321:522–525(1986). Another reference describes rodent CDRs supported byrecombinantly veneered rodent framework regions. See, for example,European Patent Publication No. 0519596. These “humanized” molecules aredesigned to minimize unwanted immunological response toward rodentanti-human antibody molecules which limits the duration andeffectiveness of therapeutic applications of those moieties in humanrecipients. For example, the antibody constant region can be engineeredsuch that it is immunologically inert (e.g., does not trigger complementlysis). See, e.g. PCT Application No. PCT/GB99/01441; UK PatentApplication No. 9809951.8. Other methods of humanizing antibodies thatmay also be utilized are disclosed by Daugherty et al., Nucl. Acids Res.19:2471–2476 (1991) and in U.S. Pat. Nos. 6,180,377; 6,054,297;5,997,867; 5,866,692; 6,210,671; and 6,350,861; and in PCT PublicationNo. WO 01/27160. Humanization can also include affinity maturation. See,e.g., U.S. Ser. No. 10/745,775, and PCT/US03/41252.

In yet another alternative, fully human antibodies may be obtained byusing commercially available mice that have been engineered to expressspecific human immunoglobulin proteins. Transgenic animals that aredesigned to produce a more desirable (e.g., fully human antibodies) ormore robust immune response may also be used for generation of humanizedor human antibodies. Examples of such technology are Xenomouse™ fromAbgenix, Inc. (Fremont, Calif.) and HuMAb-Mouse® and TC Mouse™ fromMedarex, Inc. (Princeton, N.J.).

In an alternative, antibodies may be made recombinantly and expressedusing any method known in the art. In another alternative, antibodiesmay be made recombinantly by phage display technology. See, for example,U.S. Pat. Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; andWinter et al., Annu. Rev. Immunol. 12:433–455 (1994). Alternatively, thephage display technology (McCafferty et al., Nature 348:552–553 (1990))can be used to produce human antibodies and antibody fragments in vitro,from immunoglobulin variable (V) domain gene repertoires fromunimmunized donors. According to this technique, antibody V domain genesare cloned in-frame into either a major or minor coat protein gene of afilamentous bacteriophage, such as M13 or fd, and displayed asfunctional antibody fragments on the surface of the phage particle.Because the filamentous particle contains a single-stranded DNA copy ofthe phage genome, selections based on the functional properties of theantibody also result in selection of the gene encoding the antibodyexhibiting those properties. Thus, the phage mimics some of theproperties of the B cell. Phage display can be performed in a variety offormats; for review see, e.g., Johnson, Kevin S. and Chiswell, David J.,Current Opinion in Structural Biology 3, 564–571 (1993). Several sourcesof V-gene segments can be used for phage display. Clackson et al.,Nature 352:624–628 (1991) isolated a diverse array of anti-oxazoloneantibodies from a small random combinatorial library of V genes derivedfrom the spleens of immunized mice. A repertoire of V genes fromunimmunized human donors can be constructed and antibodies to a diversearray of antigens (including self-antigens) can be isolated essentiallyfollowing the techniques described by Mark et al., J. Mol. Biol.222:581–597 (1991), or Griffith et al., EMBO J. 12:725–734 (1993). In anatural immune response, antibody genes accumulate mutations at a highrate (somatic hypermutation). Some of the changes introduced will conferhigher affinity, and B cells displaying high-affinity surfaceimmunoglobulin are preferentially replicated and differentiated duringsubsequent antigen challenge. This natural process can be mimicked byemploying the technique known as “chain shuffling.” Marks, et al.,Bio/Technol. 10:779–783 (1992)). In this method, the affinity of“primary” human antibodies obtained by phage display can be improved bysequentially replacing the heavy and light chain V region genes withrepertoires of naturally occurring variants (repertoires) of V domaingenes obtained from unimmunized donors. This technique allows theproduction of antibodies and antibody fragments with affinities in thepM–nM range. A strategy for making very large phage antibody repertoires(also known as “the mother-of-all libraries”) has been described byWaterhouse et al., Nucl. Acids Res. 21:2265–2266 (1993). Gene shufflingcan also be used to derive human antibodies from rodent antibodies,where the human antibody has similar affinities and specificities to thestarting rodent antibody. According to this method, which is alsoreferred to as “epitope imprinting”, the heavy or light chain V domaingene of rodent antibodies obtained by phage display technique isreplaced with a repertoire of human V domain genes, creatingrodent-human chimeras. Selection on antigen results in isolation ofhuman variable regions capable of restoring a functional antigen-bindingsite, i.e., the epitope governs (imprints) the choice of partner. Whenthe process is repeated in order to replace the remaining rodent Vdomain, a human antibody is obtained (see PCT Publication No. WO93/06213, published Apr. 1, 1993). Unlike traditional humanization ofrodent antibodies by CDR grafting, this technique provides completelyhuman antibodies, which have no framework or CDR residues of rodentorigin.

It is apparent that although the above discussion pertains to humanizedantibodies, the general principles discussed are applicable tocustomizing antibodies for use, for example, in dogs, cats, primate,equines and bovines. It is further apparent that one or more aspects ofhumanizing an antibody described herein may be combined, e.g., CDRgrafting, framework mutation and CDR mutation.

Antibodies may be made recombinantly by first isolating the antibodiesand antibody producing cells from host animals, obtaining the genesequence, and using the gene sequence to express the antibodyrecombinantly in host cells (e.g., CHO cells). Another method which maybe employed is to express the antibody sequence in plants (e.g.,tobacco) or transgenic milk. Methods for expressing antibodiesrecombinantly in plants or milk have been disclosed. See, for example,Peeters, et al. Vaccine 19:2756 (2001); Lonberg, N. and D. Huszar Int.Rev. Immunol 13:65 (1995); and Pollock, et al., J Immunol Methods231:147(1999). Methods for making derivatives of antibodies, e.g.,humanized, single chain, etc. are known in the art.

Immunoassays and flow cytometry sorting techniques such as fluorescenceactivated cell sorting (FACS) can also be employed to isolate antibodiesthat are specific for NGF.

The antibodies can be bound to many different carriers. Carriers can beactive and/or inert. Examples of well-known carriers includepolypropylene, polystyrene, polyethylene, dextran, nylon, amylases,glass, natural and modified celluloses, polyacrylamides, agaroses andmagnetite. The nature of the carrier can be either soluble or insolublefor purposes of the invention. Those skilled in the art will know ofother suitable carriers for binding antibodies, or will be able toascertain such, using routine experimentation.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the monoclonal antibodies). The hybridoma cells serve asa preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors (such as expression vectors disclosed in PCTPublication No. WO 87/04462), which are then transfected into host cellssuch as E. coli cells, simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. See, e.g., PCT Publication No. WO 87/04462. TheDNA also may be modified, for example, by substituting the codingsequence for human heavy and light chain constant domains in place ofthe homologous murine sequences, Morrison et al., Proc. Nat. Acad. Sci.81:6851 (1984), or by covalently joining to the immunoglobulin codingsequence all or part of the coding sequence for a non-immunoglobulinpolypeptide. In that manner, “chimeric” or “hybrid” antibodies areprepared that have the binding specificity of an anti-NGF monoclonalantibody herein.

Anti-NGF antibodies may be characterized using methods well known in theart. For example, one method is to identify the epitope to which itbinds, or “epitope mapping.” There are many methods known in the art formapping and characterizing the location of epitopes on proteins,including solving the crystal structure of an antibody-antigen complex,competition assays, gene fragment expression assays, and syntheticpeptide-based assays, as described, for example, in Chapter 11 of Harlowand Lane, Using Antibodies, a Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1999. In an additionalexample, epitope mapping can be used to determine the sequence to whichan anti-NGF antibody binds. Epitope mapping is commercially availablefrom various sources, for example, Pepscan Systems (Edelhertweg 15, 8219PH Lelystad, The Netherlands). The epitope can be a linear epitope,i.e., contained in a single stretch of amino acids, or a conformationalepitope formed by a three-dimensional interaction of amino acids thatmay not necessarily be contained in a single stretch (primary structurelinear sequence). Peptides of varying lengths (e.g., at least 4–6 aminoacids long) can be isolated or synthesized (e.g., recombinantly) andused for binding assays with an anti-NGF antibody. In another example,the epitope to which the anti-NGF antibody binds can be determined in asystematic screening by using overlapping peptides derived from the NGFsequence and determining binding by the anti-NGF antibody. According tothe gene fragment expression assays, the open reading frame encoding NGFis fragmented either randomly or by specific genetic constructions andthe reactivity of the expressed fragments of NGF with the antibody to betested is determined. The gene fragments may, for example, be producedby PCR and then transcribed and translated into protein in vitro, in thepresence of radioactive amino acids. The binding of the antibody to theradioactively labeled NGF fragments is then determined byimmunoprecipitation and gel electrophoresis. Certain epitopes can alsobe identified by using large libraries of random peptide sequencesdisplayed on the surface of phage particles (phage libraries).Alternatively, a defined library of overlapping peptide fragments can betested for binding to the test antibody in simple binding assays. In anadditional example, mutagenesis of an antigen binding domain, domainswapping experiments and alanine scanning mutagenesis can be performedto identify residues required, sufficient, and/or necessary for epitopebinding. For example, domain swapping experiments can be performed usinga mutant NGF in which various fragments of the NGF polypeptide have beenreplaced (swapped) with sequences from a closely related, butantigenically distinct protein (such as another member of theneurotrophin protein family). By assessing binding of the antibody tothe mutant NGF, the importance of the particular NGF fragment toantibody binding can be assessed.

Yet another method which can be used to characterize an anti-NGFantibody is to use competition assays with other antibodies known tobind to the same antigen, i.e., various fragments on NGF, to determineif the anti-NGF antibody binds to the same epitope as other antibodies.Competition assays are well known to those of skill in the art. Exampleof antibodies that can be used in the competition assays for the presentinvention include MAb 911, 912, 938, as described in Hongo, et al.,Hybridoma 19:215–227 (2000).

Other NGF Antagonists

NGF antagonists other than anti-NGF antibodies may be used. In someembodiments of the invention, the NGF antagonist comprises at least oneantisense molecule capable of blocking or decreasing the expression of afunctional NGF. Nucleotide sequences of the NGF are known and arereadily available from publicly available databases. See, e.g., Borsaniet al., Nuc. Acids Res. 1990, 18, 4020; Accession Number NM 002506;Ullrich et al., Nature 303:821–825 (1983). It is routine to prepareantisense oligonucleotide molecules that will specifically bind NGF MRNAwithout cross-reacting with other polynucleotides. Exemplary sites oftargeting include, but are not limited to, the initiation codon, the 5′regulatory regions, the coding sequence and the 3′ untranslated region.In some embodiments, the oligonucleotides are about 10 to 100nucleotides in length, about 15 to 50 nucleotides in length, about 18 to25 nucleotides in length, or more. The oligonucleotides can comprisebackbone modifications such as, for example, phosphorothioate linkages,and 2′-O sugar modifications well know in the art. Exemplary antisensemolecules include the NGF antisense molecules described in U.S.Publication No. 20010046959; see also http://www.rna-tec.com/repair.htm.

In other embodiments, the NGF antagonist comprises at least oneantisense molecule capable of blocking or decreasing the expression of afunctional NGF receptor (such as TrkA and/or p75). Woolf et al., J.Neurosci. (2001) 21(3):1047–55; Taglialetela et al, J Neurochem (1996)66(5): 1826–35. Nucleotide sequences of TrkA and p75 are known and arereadily available from publicly available databases.

Alternatively, NGF expression and/or release and/or NGF receptorexpression can be decreased using gene knockdown, morpholinooligonucleotides, RNAi, or ribozymes, methods that are well-known in theart. See

-   http://www.macalester.edu/˜montgomery/RNAi.html;-   http://pub32.ezboard.com/fmorpholinosfrm19.showMessage?topicID=6.topic;-   http://www.highveld.com/ribozyme.html.

In other embodiments, the NGF antagonist comprises at least one NGFinhibitory compound. As used herein, “NGF inhibitory compound” refers toa compound other than an anti-NGF antibody that directly or indirectlyreduces, inhibits, neutralizes, or abolishes NGF biological activity. AnNGF inhibitory compound should exhibit any one or more of the followingcharacteristics: (a) bind to NGF and inhibit NGF biological activityand/or downstream pathways mediated by NGF signaling function; (b)prevent, ameliorate, or treat any aspect of bone cancer pain includingcancer pain associated with bone metastasis; (c) block or decrease NGFreceptor activation (including TrkA receptor dimerization and/orautophosphorylation); (d) increase clearance of NGF; (e) inhibit(reduce) NGF synthesis, production or release. Exemplary NGF inhibitorycompounds include the small molecule NGF inhibitors described in U.S.Publication No. 20010046959; the compounds that inhibit NGF's binding top75, as described in PCT Publication No. WO 00/69829, and PD90780[7-(benzolylamino)-4,9-dihydro-4-methyl-9-oxo-pyrazolo[5,1-b]quinazoline-2-carboxylic acid] as described by Colquhoun et al., J.Pharmacol. Exp. Ther. 310(2):505–11 (2004); the compounds that inhibitNGF's binding to TrkA and/or p75, as described in PCT Publication No. WO98/17278. Additional examples of NGF inhibitory compounds include thecompounds described in PCT Publication Nos. WO 02/17914 and WO 02/20479,and in U.S. Pat. Nos. 5,342,942; 6,127,401; and 6,359,130. Furtherexemplary NGF inhibitory compounds are compounds that are competitiveinhibitors of NGF. See U.S. Pat. No. 6,291,247. Furthermore, one skilledin the art can prepare other small molecules NGF inhibitory compounds.

In some embodiments, an NGF inhibitory compound binds NGF. Exemplarysites of targeting (binding) include, but are not limited to, theportion of the NGF that binds to the TrkA receptor and/or p75 receptor,and those portions of the NGF that are adjacent to the receptor-bindingregion and which are responsible, in part, for the correctthree-dimensional shape of the receptor-binding portion. In anotherembodiment, an NGF inhibitory compound binds an NGF receptor (such asTrkA and/or p75) and inhibits an NGF biological activity. Exemplarysites of targeting include those portions of TrkA and/or p75 that bindto NGF.

In embodiments comprising small molecules, a small molecule can have amolecular weight of about any of 100 to 20,000 daltons, 500 to 15,000daltons, or 1000 to 10,000 daltons. Libraries of small molecules arecommercially available. The small molecules can be administered usingany means known in the art, including inhalation, intraperitoneally,intravenously, intramuscularly, subcutaneously, intrathecally,intraventricularly, orally, enterally, parenterally, intranasally, ordermally. In general, when the NGF-antagonist according to the inventionis a small molecule, it will be administered at the rate of 0.1 to 300mg/kg of the weight of the patient divided into one to three or moredoses. For an adult patient of normal weight, doses ranging from 1 mg to5 g per dose can be administered.

In other embodiments, the NGF antagonist comprises at least one NGFstructural analog. “NGF structural analogs” in the present inventionrefer to compounds that have a similar 3-dimensional structure as partof that of NGF and which bind to an NGF receptor under physiologicalconditions in vitro or in vivo, wherein the binding at least partiallyinhibits an NGF biological activity. In one embodiment, the NGFstructural analog binds to a TrkA and/or a p75 receptor. Exemplary NGFstructural analogs include, but are not limited to, the bicyclicpeptides described in PCT Publication No. WO 97/15593; the bicyclicpeptides described in U.S. Pat. No. 6,291,247; the cyclic compoundsdescribed in U.S. Pat. No. 6,017,878; and NGF-derived peptides describedin PCT Publication No. WO 89/09225. Suitable NGF structural analogs canalso be designed and synthesized through molecular modeling ofNGF-receptor binding, for example by the method described in PCTPublication No. WO 98/06048. The NGF structural analogs can be monomersor dimers/oligomers in any desired combination of the same or differentstructures to obtain improved affinities and biological effects.

In other embodiments, the invention provides an NGF antagonistcomprising at least one dominant-negative mutant of the TrkA receptorand/or p75 receptor. One skilled in the art can preparedominant-negative mutants of, e.g., the TrkA receptor such that thereceptor will bind the NGF and, thus, act as a “sink” to capture NGFs.The dominant-negative mutants, however, will not have the normalbioactivity of the TrkA receptor upon binding to NGF. Exemplarydominant-negative mutants include, but are not limited to, the mutantsdescribed in the following references: Li et al., Proc. Natl. Acad. Sci.USA 1998, 95, 10884; Eide et al., J. Neurosci. 1996, 16, 3123; Liu etal., J. Neurosci 1997, 17, 8749; Klein et al., Cell 1990, 61, 647;Valenzuela et al., Neuron 1993, 10, 963; Tsoulfas et al., Neuron 1993,10, 975; and Lamballe et al., EMBO J. 1993, 12, 3083, each of which isincorporated herein by reference in its entirety. The dominant negativemutants can be administered in protein form or in the form of anexpression vector such that the dominant negative mutant, e.g., mutantTrkA receptor, is expressed in vivo. The protein or expression vectorcan be administered using any means known in the art, such asintraperitoneally, intravenously, intramuscularly, subcutaneously,intrathecally, intraventricularly, orally, enterally, parenterally,intranasally, dermally, or by inhalation. For example, administration ofexpression vectors includes local or systemic administration, includinginjection, oral administration, particle gun or catheterizedadministration, and topical administration. One skilled in the art isfamiliar with administration of expression vectors to obtain expressionof an exogenous protein in vivo. See, e.g., U.S. Pat. Nos. 6,436,908;6,413,942; and 6,376,471.

Targeted delivery of therapeutic compositions containing an antisensepolynucleotide, expression vector, or subgenomic polynucleotides canalso be used. Receptor-mediated DNA delivery techniques are describedin, for example, Findeis et al., Trends Biotechnol. (1993) 11:202; Chiouet al., Gene Therapeutics: Methods And Applications Of Direct GeneTransfer (J. A. Wolff, ed.) (1994); Wu et al., J. Biol. Chem. (1988)263:621; Wu et al., J. Biol. Chem. (1994) 269:542; Zenke et al., Proc.Natl. Acad. Sci. USA (1990) 87:3655; Wu et al., J. Biol. Chem. (1991)266:338. Therapeutic compositions containing a polynucleotide areadministered in a range of about 100 ng to about 200 mg of DNA for localadministration in a gene therapy protocol. In some embodiments,concentration ranges of about 500 ng to about 50 mg, about 1 μg to about2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNAor more can also be used during a gene therapy protocol. The therapeuticpolynucleotides and polypeptides of the present invention can bedelivered using gene delivery vehicles. The gene delivery vehicle can beof viral or non-viral origin (see generally, Jolly, Cancer Gene Therapy(1994) 1:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, HumanGene Therapy (1995) 1:185; and Kaplitt, Nature Genetics (1994) 6:148).Expression of such coding sequences can be induced using endogenousmammalian or heterologous promoters and/or enhancers. Expression of thecoding sequence can be either constitutive or regulated.

Viral-based vectors for delivery of a desired polynucleotide andexpression in a desired cell are well known in the art. Exemplaryviral-based vehicles include, but are not limited to, recombinantretroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622;WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S.Pat. Nos. 5, 219,740 and 4,777,127; GB Patent No. 2,200,651; and EPPatent No. 0 345 242), alphavirus-based vectors (e.g., Sindbis virusvectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross Rivervirus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitisvirus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), andadeno-associated virus (AAV) vectors (see, e.g., PCT Publication Nos. WO94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO95/00655). Administration of DNA linked to killed adenovirus asdescribed in Curiel, Hum. Gene Ther. (1992) 3:147 can also be employed.

Non-viral delivery vehicles and methods can also be employed, including,but not limited to, polycationic condensed DNA linked or unlinked tokilled adenovirus alone (see, e.g., Curiel, Hum. Gene Ther. (1992)3:147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989)264:16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S.Pat. No. 5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO95/30763; and WO 97/42338) and nucleic charge neutralization or fusionwith cell membranes. Naked DNA can also be employed. Exemplary naked DNAintroduction methods are described in PCT Publication No. WO 90/11092and U.S. Pat. No. 5,580,859. Liposomes that can act as gene deliveryvehicles are described in U.S. Pat. No. 5,422,120; PCT Publication Nos.WO 95/13796; WO 94/23697; WO 91/14445; and EP Patent No. 0524968.Additional approaches are described in Philip, Mol. Cell Biol. (1994)14:2411, and in Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

It is also apparent that an expression vector can be used to directexpression of any of the protein-based NGF antagonists described herein(e.g., anti-NGF antibody, TrkA immunoadhesin, etc.). For example, otherTrkA receptor fragments that are capable of blocking (from partial tocomplete blocking) NGF and/or an NGF biological activity are known inthe art.

In another embodiment, the NGF antagonist comprises at least one TrkAimmunoadhesin. TrkA immunoadhesins as used herein refer to solublechimeric molecules comprising the extracellular domain of a TrkAreceptor and an immunoglobulin sequence, which retains the bindingspecificity of the TrkA receptor (substantially retains the bindingspecificity of the trkA receptor) and is capable of binding to NGF.

TrkA immunoadhesins are known in the art, and have been found to blockthe binding of NGF to the TrkA receptor. See, e.g., U.S. Pat. No.6,153,189. Brennan et al. report administration of TrkA immunoadhesin ina rat model of post-surgical pain. See Society for NeuroscienceAbstracts 24 (1–2) 880 (1998). In one embodiment, the TrkA immunoadhesincomprises a fusion of a TrkA receptor amino acid sequence (or a portionthereof) from TrkA extracellular domain capable of binding NGF (in someembodiments, an amino acid sequence that substantially retains thebinding specificity of the trkA receptor) and an immunoglobulinsequence. In some embodiments, the TrkA receptor is a human TrkAreceptor sequence, and the fusion is with an immunoglobulin constantdomain sequence. In other embodiments, the immunoglobulin constantdomain sequence is an immunoglobulin heavy chain constant domainsequence. In other embodiments, the association of two TrkAreceptor-immunoglobulin heavy chain fusions (e.g., via covalent linkageby disulfide bond(s)) results in a homodimeric immunoglobulin-likestructure. An immunoglobulin light chain can further be associated withone or both of the TrkA receptor-immunoglobulin chimeras in thedisulfide-bonded dimer to yield a homotrimeric or homotetramericstructure. Examples of suitable TrkA immunoadhesins include thosedescribed in U.S. Pat. No. 6,153,189.

In another embodiment, the NGF antagonist comprises at least oneanti-TrkA antibody capable of blocking, suppressing, altering, and/orreducing NGF physical interaction with the TrkA receptor and/ordownstream signaling, whereby an NGF biological activity is reducedand/or blocked. Anti-TrkA antibodies are known in the art. Exemplaryanti-TrkA antibodies include those described in PCT Publication Nos. WO97/21732, WO 00/73344, WO 02/15924, and U.S. Publication No.20010046959.

In another embodiment, the NGF antagonist comprises at least oneanti-p75 antibody capable of blocking, suppressing and/or reducing NGFphysical interaction with the p75 receptor and/or downstream signaling,whereby an NGF biological activity is reduced and/or blocked.

In another embodiment, the NGF antagonist comprises at least one kinaseinhibitor capable of inhibiting downstream kinase signaling associatedwith TrkA and/or p75 receptor activity. An exemplary kinase inhibitor isK252a or K252b, which is known in the art and described in Knusel etal., J. Neurochem. 59:715–722 (1992); Knusel et al., J. Neurochemistry57:955–962 (1991); Koizumi et al., J. Neuroscience 8:715–721 (1988);Hirata et al., Chemical Abstracts 111:728, XP00204135, see abstract and12th Collective Chemical Substance Index, p. 34237, c. 3 (5–7), 55–60,66–69), p. 34238, c.1 (41–44), c.2 (25–27, 32–33), p. 3423, c.3 (48–50,52–53); and U.S. Pat. No. 6,306,849.

It is expected that a number of other categories of NGF antagonists willbe identified if sought for by the clinician.

Identification of NGF Antagonists

Anti-NGF antibodies and other NGF antagonists can be identified orcharacterized using methods known in the art, whereby reduction,amelioration, or neutralization of an NGF biological activity isdetected and/or measured. Methods described in PCT WO 04/065560 can beused. Another method, for example, a kinase receptor activation (KIRA)assay described in U.S. Pat. Nos. 5,766,863 and 5,891,650, can be usedto identify NGF antagonists. This ELISA-type assay is suitable forqualitative or quantitative measurement of kinase activation bymeasuring the autophosphorylation of the kinase domain of a receptorprotein tyrosine kinase (hereinafter “rPTK”), e.g. TrkA receptor, aswell as for identification and characterization of potential antagonistsof a selected rPTK, e.g., TrkA. The first stage of the assay involvesphosphorylation of the kinase domain of a kinase receptor, for example,a TrkA receptor, wherein the receptor is present in the cell membrane ofa eukaryotic cell. The receptor may be an endogenous receptor or nucleicacid encoding the receptor, or a receptor construct, may be transformedinto the cell. Typically, a first solid phase (e.g., a well of a firstassay plate) is coated with a substantially homogeneous population ofsuch cells (usually a mammalian cell line) so that the cells adhere tothe solid phase. Often, the cells are adherent and thereby adherenaturally to the first solid phase. If a “receptor construct” is used,it usually comprises a fusion of a kinase receptor and a flagpolypeptide. The flag polypeptide is recognized by the capture agent,often a capture antibody, in the ELISA part of the assay. An analyte,such as a candidate anti-NGF antibody or other NGF antagonists, is thenadded together with NGF to the wells having the adherent cells, suchthat the tyrosine kinase receptor (e.g. TrkA receptor) is exposed to (orcontacted with) NGF and the analyte. This assay enables identificationof antibodies (or other NGF antagonists) that inhibit activation of TrkAby its ligand NGF. Following exposure to NGF and the analyte, theadhering cells are solubilized using a lysis buffer (which has asolubilizing detergent therein) and gentle agitation, thereby releasingcell lysate which can be subjected to the ELISA part of the assaydirectly, without the need for concentration or clarification of thecell lysate.

The cell lysate thus prepared is then ready to be subjected to the ELISAstage of the assay. As a first step in the ELISA stage, a second solidphase (usually a well of an ELISA microtiter plate) is coated with acapture agent (often a capture antibody) which binds specifically to thetyrosine kinase receptor, or, in the case of a receptor construct, tothe flag polypeptide. Coating of the second solid phase is carried outso that the capture agent adheres to the second solid phase. The captureagent is generally a monoclonal antibody, but, as is described in theexamples herein, polyclonal antibodies may also be used. The cell lysateobtained is then exposed to, or contacted with, the adhering captureagent so that the receptor or receptor construct adheres to (or iscaptured in) the second solid phase. A washing step is then carried out,so as to remove unbound cell lysate, leaving the captured receptor orreceptor construct. The adhering or captured receptor or receptorconstruct is then exposed to, or contacted with, an anti-phosphotyrosineantibody which identifies phosphorylated tyrosine residues in thetyrosine kinase receptor. In one embodiment, the anti-phosphotyrosineantibody is conjugated (directly or indirectly) to an enzyme whichcatalyses a color change of a non-radioactive color reagent.Accordingly, phosphorylation of the receptor can be measured by asubsequent color change of the reagent. The enzyme can be bound to theanti-phosphotyrosine antibody directly, or a conjugating molecule (e.g.,biotin) can be conjugated to the anti-phosphotyrosine antibody and theenzyme can be subsequently bound to the anti-phosphotyrosine antibodyvia the conjugating molecule. Finally, binding of theanti-phosphotyrosine antibody to the captured receptor or receptorconstruct is measured, e.g., by a color change in the color reagent.

The NGF antagonists can also be identified by incubating a candidateagent with NGF and monitoring any one or more of the followingcharacteristics: (a) binding to NGF and inhibiting NGF biologicalactivity and/or downstream pathways mediated by NGF signaling function;(b) preventing, ameliorating, or treating any aspect of bone cancer painincluding cancer pain associated with bone metastasis; (c) blocking ordecreasing NGF receptor activation (including TrkA receptor dimerizationand/or autophosphorylation); (d) increasing clearance of NGF; (e)inhibiting (reducing) NGF synthesis, production or release. In someembodiments, an NGF antagonist is identified by incubating a candidateagent with NGF and monitoring binding and attendant reduction orneutralization of a biological activity of NGF. The binding assay may beperformed with purified NGF polypeptide(s), or with cells naturallyexpressing, or transfected to express, NGF polypeptide(s). In oneembodiment, the binding assay is a competitive binding assay, where theability of a candidate antibody to compete with a known NGF antagonistfor NGF binding is evaluated. The assay may be performed in variousformats, including the ELISA format. In other embodiments, an NGFantagonist is identified by incubating a candidate agent with NGF andmonitoring attendant inhibition of TrkA receptor dimerization and/orautophosphorylation.

Following initial identification, the activity of a candidate anti-NGFantagonist can be further confirmed and refined by bioassays, known totest the targeted biological activities. Alternatively, bioassays can beused to screen candidates directly. For example, NGF promotes a numberof morphologically recognizable changes in responsive cells. Theseinclude, but are not limited to, promoting the differentiation of PC12cells and enhancing the growth of neurites from these cells (Greene andTischler, Proc. Nat. Acad Sci. USA 73:2424–2428 (1976); Urfer et al.,Biochem. 36:4775–4781 (1997); Tsoulfas et al., Neuron 10:975–990(1993)), promoting neurite outgrowth from explants of responsive sensoryand sympathetic ganglia (Levi-Montalcini, R. and Angeletti, P. Nervegrowth factor. Physiol. Rev. 48, 534–569, 1968) and promoting thesurvival of NGF dependent neurons such as embryonic dorsal rootganglion, trigeminal ganglion, or sympathetic ganglion neurons (e.g.,Chun & Patterson, Dev. Biol. 75:705–711, (1977); Buchman & Davies,Development 118:989–1001, (1993). Thus, the assay for inhibition of NGFbiological activity entail culturing NGF responsive cells with NGF plusan analyte, such as a candidate anti-NGF antibody or a candidate NGFantagonist. After an appropriate time the cell response will be assayed(cell differentiation, neurite outgrowth or cell survival).

The ability of a candidate NGF antagonist to block or neutralize abiological activity of NGF can also be assessed by monitoring theability of the candidate agent to inhibit NGF mediated survival in theembryonic rat dorsal root ganglia survival bioassay as described inHongo et al., Hybridoma 19:215–227 (2000).

Compositions for Use in the Methods of the Invention

The compositions used in the methods of the invention comprise aneffective amount of an NGF antagonist (such as anti-NGF antibody), and,in some embodiments, further comprise a pharmaceutically acceptableexcipient. In some embodiments, the composition is for use in any of themethods described herein. Examples of such compositions, as well as howto formulate, are also described in an earlier section and below. In oneembodiment, the composition comprises an NGF antagonist. In anotherembodiment, the composition comprises one or more NGF antagonists. Inanother embodiment, the composition comprises one or more NGFantagonists selected from any one or more of the following: anantagonist (e.g., an antibody) that binds (physically interacts with)NGF, an antagonist that binds to an NGF receptor (such as a TrkA and/orp75 receptor), and an antagonist that reduces (impedes and/or blocks)downstream NGF receptor signaling. In still other embodiments, thecomposition comprises any NGF antagonist that is not a TrkAimmunoadhesin (i.e., is other than a TrkA immunoadhesin). In otherembodiments, the composition comprises any NGF antagonist that is otherthan an anti-NGF antibody. In still other embodiments, the compositioncomprises any NGF antagonist that is other than a TrkA immunoadhesin andother than an anti-NGF antibody. In other embodiments, an NGF antagonistinhibits (reduces) NGF synthesis, production or release. In someembodiments, the NGF antagonist binds NGF and does not significantlycross-react with related neurotrophins (such as NT3, NT4/5, and/orBDNF). In some embodiments, the NGF antagonist is not associated with anadverse immune response. In some embodiments, the NGF antagonist isselected from the group consisting of an anti-NGF antibody, ananti-sense molecule directed to an NGF (including an anti-sense moleculedirected to a nucleic acid encoding NGF), an anti-sense moleculedirected to an NGF receptor (such as TrkA and/or p75), an NGF inhibitorycompound, an NGF structural analog, a dominant-negative mutation of aTrkA receptor that binds an NGF, a TrkA immunoadhesin, an anti-TrkAantibody, an anti-p75 antibody and a kinase inhibitor. In anotherembodiment, the NGF antagonist is an anti-NGF antibody. In otherembodiments, the anti-NGF antibody recognizes human NGF. In someembodiments, the anti-NGF antibody is human. In still other embodiments,the anti-NGF antibody is humanized (such as antibody E3 describedherein). In still other embodiment, the anti-NGF antibody comprises aconstant region that does not trigger an unwanted or undesirable immuneresponse, such as antibody-mediated lysis or ADCC. In other embodiments,the anti-NGF antibody comprises one or more CDR(s) of antibody E3 (suchas one, two, three, four, five, or, in some embodiments, all six CDRsfrom E3).

It is understood that the compositions can comprise more than one NGFantagonist. For example, a composition can comprise more than one memberof a class of NGF antagonist (e.g., a mixture of anti-NGF antibodiesthat recognize different epitopes of NGF), as well as members ofdifferent classes of NGF antagonists (e.g., an anti-NGF antibody and anNGF inhibitory compound). Other exemplary compositions comprise morethan one anti-NGF antibodies that recognize the same epitope(s),different species of anti-NGF antibodies that bind to different epitopesof NGF, or different NGF inhibitory compounds.

The composition used in the present invention can further comprisepharmaceutically acceptable carriers, excipients, or stabilizers(Remington: The Science and Practice of Pharmacy 20th Ed. (2000)Lippincott Williams and Wilkins, Ed. K. E. Hoover.), in the form oflyophilized formulations or aqueous solutions. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations used, and may comprise buffers such as phosphate,citrate, and other organic acids; antioxidants including ascorbic acidand methionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrans; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG). Pharmaceutically acceptable excipients arefurther described herein.

The NGF antagonist and compositions thereof can also be used inconjunction with other agents that serve to enhance and/or complementthe effectiveness of the agents. In some embodiments, the other agent isan opioid analgesic. In some embodiments, the other agent is an NSAID.In some embodiments, these agents are not opioid analgesics. In someembodiments, these agents are not NSAID.

Kits

The invention also provides kits for use in the instant methods. Kits ofthe invention include one or more containers comprising an NGFantagonist (such as an antibody, such as humanized antibody E3 describedherein), and in some embodiments, further comprise instructions for usein accordance with any of the methods of the invention described herein.In some embodiments, the NGF antagonist is any NGF antagonist describedherein. In still other embodiments, the kit comprises an NGF antagonistthat is not a TrkA immunoadhesin (i.e., is other than a TrkAimmunoadhesin). In other embodiments, the kit comprises an NGFantagonist that is other than an anti-NGF antibody. In still otherembodiments, the kit comprises any NGF antagonist that is other than aTrkA immunoadhesin and other than an anti-NGF antibody. In someembodiment, the kit comprises an anti-NGF antibody (such as antibody E3described herein). In other embodiments, the kit comprises an anti-NGFantibody comprising one or more CDR(s) of antibody E3 (such as one, two,three, four, five, or, in some embodiments, all six CDRs from E3). Insome embodiments, the kit includes an opioid analgesic. In someembodiments, the kit includes an NSAID. In some embodiments, the kitdoes not include an opioid analgesic. In some embodiments, the kit doesnot include an NSAID. In some embodiments, the included instructionscomprise a description of administration of the NGF antagonist to treat,ameliorate or prevent bone cancer pain including cancer pain associatedwith bone metastasis according to any of the methods described herein.The kit may further comprise a description of selecting an individualsuitable for treatment based on identifying whether that individual hasbone cancer pain including cancer pain associated with bone metastasisor whether the individual is at risk of bone cancer pain includingcancer pain associated with bone metastasis. In still other embodiments,the instruction comprises a description of administering an NGFantagonist to treat, prevent and/or ameliorate bone cancer painincluding cancer pain associated with bone metastasis. In still otherembodiments, the instructions comprise a description of administering anNGF antagonist to an individual at risk of bone cancer pain includingcancer pain associated with bone metastasis. In some embodiments, theNGF antagonist is co-administered with an opioid analgesic. In someembodiments, the NGF antagonist is co-administered with an NSAID. Insome embodiments, the NGF antagonist is co-administered with an opioidanalgesic and an NSAID. In some embodiments, the NGF antagonist is notco-administered with an opioid analgesic. In some embodiments, the NGFantagonist is not co-administered with an NSAID.

The instructions relating to the use of an NGF antagonist generallyinclude information as to dosage, dosing schedule, and route ofadministration for the intended treatment. The containers may be unitdoses, bulk packages (e.g., multi-dose packages) or sub-unit doses.Instructions supplied in the kits of the invention are typically writteninstructions on a label or package insert (e.g., a paper sheet includedin the kit), but machine-readable instructions (e.g., instructionscarried on a magnetic or optical storage disk) are also acceptable.

The label or package insert indicates that the composition is used fortreating, ameliorating and/or preventing bone cancer pain includingcancer pain associated with bone metastasis. Instructions may beprovided for practicing any of the methods described herein.

The kits of this invention are in suitable packaging. Suitable packagingincludes, but is not limited to, vials, bottles, jars, flexiblepackaging (e.g., sealed Mylar or plastic bags), and the like. Alsocontemplated are packages for use in combination with a specific device,such as an inhaler, nasal administration device (e.g., an atomizer) oran infusion device such as a minipump. A kit may have a sterile accessport (for example the container may be an intravenous solution bag or avial having a stopper pierceable by a hypodermic injection needle). Thecontainer may also have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an NGF antagonist, such as an anti-NGF antibody. Thecontainer may further comprise a second pharmaceutically active agent.

Kits may optionally provide additional components such as buffers andinterpretive information. Normally, the kit comprises a container and alabel or package insert(s) on or associated with the container.

In some embodiments, the invention provides articles of manufacturecomprising contents of the kits described above. In some embodiments,the kits comprise an NGF antagonist (such as anti-NGF antibody) withinformation indicating use to treat bone cancer pain including cancerpain associate with bone metastasis.

Administration of an NGF Antagonist and Assessment of Treatment

The NGF antagonist can be administered to an individual via any suitableroute. For example, the NGF antagonist can be administered orally,intravenously, sublingually, subcutaneously, intraarterially,intrasynovially, intravescicular (such as via the bladder),intramuscularly, intracardiacly, intrathoracicly, intraperitoneally,intraventricularly, sublingually, by inhalation, by suppository, andtransdermally. They can be administered orally, for example, in the formof tablets, troches, capsules, elixirs, suspensions, syrups, wafers,lollypops, chewing gum or the like prepared by art recognizedprocedures. It should be apparent to a person skilled in the art thatthe examples described herein are not intended to be limiting but to beillustrative of the techniques available.

Accordingly, in some embodiments, the NGF antagonist, such as ananti-NGF antibody, is administered to a individual in accordance withknown methods, such as intravenous administration, e.g., as a bolus orby continuous infusion over a period of time, by intramuscular,intraperitoneal, intracerebrospinal, subcutaneous, intra-articular,intrasynovial, intrathecal, oral, inhalation or topical routes.Commercially available nebulizers for liquid formulations, including jetnebulizers and ultrasonic nebulizers are useful for administration.Liquid formulations can be directly nebulized and lyophilized powder canbe nebulized after reconstitution. Alternatively, NGF antagonist can beaerosolized using a fluorocarbon formulation and a metered dose inhaler,or inhaled as a lyophilized and milled powder.

In one embodiment, an NGF antagonist is administered via site-specificor targeted local delivery techniques. Examples of site-specific ortargeted local delivery techniques include various implantable depotsources of the NGF antagonist or local delivery catheters, such asinfusion catheters, an indwelling catheter, or a needle catheter,synthetic grafts, adventitial wraps, shunts and stents or otherimplantable devices, site specific carriers, direct injection, or directapplication. See, e.g., PCT Publication No. WO 00/53211 and U.S. Pat.No. 5,981,568.

Various formulations of an NGF antagonist (such as anti-NGF antibody)may be used for administration. In some embodiments, an NGF antagonistmay be administered neat. In some embodiments, the NGF antagonistcomprises an anti-NGF antibody, and may be in various formulations,including formulations comprising a pharmaceutically acceptableexcipient. Pharmaceutically acceptable excipients are known in the art,and are relatively inert substances that facilitate administration of apharmacologically effective substance. For example, an excipient cangive form or consistency, or act as a diluent. Suitable excipientsinclude, but are not limited to, stabilizing agents, wetting andemulsifying agents, salts for varying osmolarity, encapsulating agents,buffers, and skin penetration enhancers. Excipients as well asformulations for parenteral and nonparenteral drug delivery are setforth in Remington, The Science and Practice of Pharmacy 20th Ed. MackPublishing (2000).

In some embodiments, these agents are formulated for administration byinjection (e.g., intraperitoneally, intravenously, subcutaneously,intramuscularly, etc.). Accordingly, these agents can be combined withpharmaceutically acceptable vehicles such as saline, Ringer's solution,dextrose solution, and the like. The particular dosage regimen, i.e.,dose, timing and repetition, will depend on the particular individualand that individual's medical history.

An anti-NGF antibody can be administered using any suitable method,including by injection (e.g., intraperitoneally, intravenously,subcutaneously, intramuscularly, etc.). Anti-NGF antibodies can also beadministered via inhalation, as described herein. Generally, foradministration of anti-NGF antibodies, an initial candidate dosage canbe about 2 mg/kg. For the purpose of the present invention, a typicaldaily dosage might range from about any of 0.1 μg/kg to 3 μg/kg to 30μg/kg to 300 μg/kg to 3 mg/kg, to 30 mg/kg to 100 mg/kg or more,depending on the factors mentioned above. For repeated administrationsover several days or longer, depending on the condition, the treatmentis sustained until a desired suppression of symptoms occurs or untilsufficient therapeutic levels are achieved to reduce cancer painassociated with bone metastasis. An exemplary dosing regimen comprisesadministering an initial dose of about 2 mg/kg, followed by a weeklymaintenance dose of about 1 mg/kg of the anti-NGF antibody, or followedby a maintenance dose of about 1 mg/kg every other week. However, otherdosage regimens may be useful, depending on the pattern ofpharmacokinetic decay that the practitioner wishes to achieve. Forexample, dosing from one-four time a week is contemplated. In someembodiments, dosing ranging from about 3 μg/mg to about 2 mg/kg (such asabout 3 μg/mg, about 10 μg/mg, about 30 μg/mg, about 100 μg/mg, about300 μg/mg, about 1 mg/kg, and about 2 mg/kg) may be used. In someembodiments, dosing frequency is once every week, every 2 weeks, every 4weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every9 weeks, or every 10 weeks; or once every month, every 2 months, orevery 3 months, or longer. The progress of this therapy is easilymonitored by conventional techniques and assays. The dosing regimen(including the NGF antagonist(s) used) can vary over time.

In general, when it is not an antibody, an NGF antagonist may (in someembodiments) be administered at the rate of about 0.1 to 300 mg/kg ofthe weight of the patient divided into one to three doses, or asdisclosed herein. In some embodiments, for an adult patient of normalweight, doses ranging from about 0.3 to 5.00 mg/kg may be administered.The particular dosage regimen, i.e., dose, timing and repetition, willdepend on the particular individual and that individual's medicalhistory, as well as the properties of the individual agents (such as thehalf-life of the agent, and other considerations well known in the art).

For the purpose of the present invention, the appropriate dosage of anNGF antagonist will depend on the NGF antagonist(s) (or compositionsthereof) employed, the type and severity of the pain to be treated,whether the agent is administered for preventive or therapeuticpurposes, previous therapy, the patient's clinical history and responseto the agent, and the discretion of the attending physician. Typicallythe clinician will administer an NGF antagonist, such as an anti-NGFantibody, until a dosage is reached that achieves the desired result.

Empirical considerations, such as the half-life, generally willcontribute to the determination of the dosage. For example, antibodiesthat are compatible with the human immune system, such as humanizedantibodies or fully human antibodies, may be used to prolong half-lifeof the antibody and to prevent the antibody being attacked by the host'simmune system. Frequency of administration may be determined andadjusted over the course of therapy, and is generally, but notnecessarily, based on treatment and/or suppression and/or ameliorationand/or delay of pain. Alternatively, sustained continuous releaseformulations of anti-NGF antibodies may be appropriate. Variousformulations and devices for achieving sustained release are known inthe art.

In one embodiment, dosages for an NGF antagonist may be determinedempirically in individuals who have been given one or moreadministration(s) of NGF antagonist (such as an antibody). Individualsare given incremental dosages of an NGF antagonist, e.g., anti-NGFantibody. To assess efficacy of an NGF antagonist, an indicator of paincan be followed.

Administration of an NGF antagonist in accordance with the method in thepresent invention can be continuous or intermittent, depending, forexample, upon the recipient's physiological condition, whether thepurpose of the administration is therapeutic or prophylactic, and otherfactors known to skilled practitioners. The administration of an NGFantagonist (for example if the NGF antagonist is an anti-NGF antibody)may be essentially continuous over a preselected period of time or maybe in a series of spaced dose, e.g., either before, during, or afterdeveloping pain; before; during; before and after; during and after;before and during; or before, during, and after developing pain.Administration can be before, during and/or after cancer hasmetastasized to bone, and any other event likely to give rise to cancerpain associated with bone metastasis.

In some embodiments, more than one NGF antagonist, such as an antibody,may be present. The antagonist can be the same or different from eachother. At least one, at least two, at least three, at least four, atleast five different NGF antagonists can be present. Generally, thoseNGF antagonists have complementary activities that do not adverselyaffect each other. NGF antagonists can also be used in conjunction withother agents that serve to enhance and/or complement the effectivenessof the agents. In some embodiments, the NGF antagonist is notco-administered with an opioid analgesic. In some embodiments, the NGFantagonist is not co-administered with an NSAID.

Therapeutic formulations of the NGF antagonist (such as an antibody)used in accordance with the present invention are prepared for storageby mixing an antibody having the desired degree of purity with optionalpharmaceutically acceptable carriers, excipients or stabilizers(Remington, The Science and Practice of Pharmacy 20th Ed. MackPublishing (2000)), in the form of lyophilized formulations or aqueoussolutions. Acceptable carriers, excipients, or stabilizers are nontoxicto recipients at the dosages and concentrations employed, and maycomprise buffers such as phosphate, citrate, and other organic acids;salts such as sodium chloride; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens, such asmethyl or propyl paraben; catechol; resorcinol; cyclohexanol;3-pentanol; and m-cresol); low molecular weight (less than about 10residues) polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

Liposomes containing the NGF antagonist (such as an antibody) areprepared by methods known in the art, such as described in Epstein, etal., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang, et al., Proc.Natl Acad. Sci. USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and4,544,545. Liposomes with enhanced circulation time are disclosed inU.S. Pat. No. 5,013,556. Particularly useful liposomes can be generatedby the reverse phase evaporation method with a lipid compositioncomprising phosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing(2000).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or ′poly(v nylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), sucrose acetate isobutyrate, andpoly-D-(−)-3-hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by, for example, filtration through sterilefiltration membranes. Therapeutic anti-NGF antibody compositions aregenerally placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having a stopper pierceableby a hypodermic injection needle.

The compositions according to the present invention may be in unitdosage forms such as tablets, pills, capsules, powders, granules,solutions or suspensions, or suppositories, for oral, parenteral orrectal administration, or administration by inhalation or insufflation.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical carrier, e.g. conventionaltableting ingredients such as corn starch, lactose, sucrose, sorbitol,talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, andother pharmaceutical diluents, e.g. water, to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention, or a non-toxic pharmaceuticallyacceptable salt thereof. When referring to these preformulationcompositions as homogeneous, it is meant that the active ingredient isdispersed evenly throughout the composition so that the composition maybe readily subdivided into equally effective unit dosage forms such astablets, pills and capsules. This solid preformulation composition isthen subdivided into unit dosage forms of the type described abovecontaining from 0.1 to about 500 mg of the active ingredient of thepresent invention. The tablets or pills of the novel composition can becoated or otherwise compounded to provide a dosage form affording theadvantage of prolonged action. For example, the tablet or pill cancomprise an inner dosage and an outer dosage component, the latter beingin the form of an envelope over the former. The two components can beseparated by an enteric layer that serves to resist disintegration inthe stomach and permits the inner component to pass intact into theduodenum or to be delayed in release. A variety of materials can be usedfor such enteric layers or coatings, such materials including a numberof polymeric acids and mixtures of polymeric acids with such materialsas shellac, cetyl alcohol and cellulose acetate.

Suitable surface-active agents include, in particular, non-ionic agents,such as polyoxyethylenesorbitans (e.g. Tween™ 20, 40, 60, 80 or 85) andother sorbitans (e.g. Span™ 20, 40, 60, 80 or 85). Compositions with asurface-active agent will conveniently comprise between 0.05 and 5%surface-active agent, and can be between 0.1 and 2.5%. It will beappreciated that other ingredients may be added, for example mannitol orother pharmaceutically acceptable vehicles, if necessary.

Suitable emulsions may be prepared using commercially available fatemulsions, such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ andLipiphysan™. The active ingredient may be either dissolved in apre-mixed emulsion composition or alternatively it may be dissolved inan oil (e.g. soybean oil, safflower oil, cottonseed oil, sesame oil,corn oil or almond oil) and an emulsion formed upon mixing with aphospholipid (e.g. egg phospholipids, soybean phospholipids or soybeanlecithin) and water. It will be appreciated that other ingredients maybe added, for example glycerol or glucose, to adjust the tonicity of theemulsion. Suitable emulsions will typically contain up to 20% oil, forexample, between 5 and 20%. The fat emulsion can comprise fat dropletsbetween 0.1 and 1.0 .μm, particularly 0.1 and 0.5 .μm, and have a pH inthe range of 5.5 to 8.0.

The emulsion compositions can be those prepared by mixing a nerve growthfactor antagonist with Intralipid™ or the components thereof (soybeanoil, egg phospholipids, glycerol and water).

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as set outabove. In some embodiments, the compositions are administered by theoral or nasal respiratory route for local or systemic effect.Compositions in preferably sterile pharmaceutically acceptable solventsmay be nebulised by use of gases. Nebulised solutions may be breatheddirectly from the nebulising device or the nebulising device may beattached to a face mask, tent or intermittent positive pressurebreathing machine. Solution, suspension or powder compositions may beadministered, preferably orally or nasally, from devices which deliverthe formulation in an appropriate manner.

Treatment efficacy can be assessed by methods well-known in the art.

EXAMPLES

The following Examples are provided to illustrate but not limit theinvention.

Example 1 Anti-NGF Monoclonal Antibody is Effective in Treating CancerPain Associated with Bone Metastasis

We used a murine bone cancer pain model to assess the efficacy oftreatment with anti-NGF antibody 911 (a mouse monoclonal antibody; seeHongo, et al., Hybridoma 19:215–227 (2000)). This murine model of bonecancer pain is developed by intramedullary injection of osteolyticsarcoma cells into the mouse femur and the needle hole is then filledwith dental amalgam to confine the tumor to bone. See Schwei et al., J.Neuroscience 19:10886–10897 (1999); and Luger et al., Pain 99:397–406(2002). Experiments were performed on adult male C3H/HeJ mice (JacksonLaboratories, Bar Harbor, Me.). On day 0, an arthrotomy was performedfollowing induction of general anesthesia with sodium pentobarbital (50mg/kg, intraperitoneal (i.p.)). A needle was inserted into the medullarycanal to create a pathway for the sarcoma cells. A depression was thenmade using a pneumatic dental high speed handpiece. In addition to naïveanimals (n=5), sham animals (n=5) were generated with an injection ofα-minimum essential media (20 μl, Sigma, St. Louis, Mo.) into theintramedullary space of the femur (designated sham) whereas sarcomaanimals (n=5 for each condition tested) were injected with mediacontaining 10⁵ 2472 osteolytic sarcoma cells (designated sarcoma orsarc) (20 μl, ATCC, Rockville, Md.). For all animals, the injection sitewas sealed with a dental amalgam plug to confine the cells or injectedmedia within the intramedullary canal and followed by irrigation withsterile water (hypotonic solution). Finally, incision closure wasachieved with wound clips. Clips were removed at day 5 so as not tointerfere with behavioral testing. A second group of sarcoma-injectedanimals was treated with anti-NGF (10 mg/kg, i.p.) on days 6 and 13.

Behavioral analysis. Animals were tested for pain-related behaviors onday 10 and day 14 post-tumor implantation Animals were behaviorallytested using the following tests: ongoing pain (spontaneous guarding andflinching); ambulatory pain (limb use and rotarod), and movement-evokedpain (palpation-evoked guarding and palpation-evoked flinching). Animalswere placed in a clear plastic observation box with a wire mesh floorand allowed to habituate for a period of 30 min. After acclimation,spontaneous guarding, spontaneous flinching, limb use during normalambulation in an open field, and guarding during forced ambulation wereassessed. Palpation-induced guarding and flinching were measured afterthe 2 min period of normally non-noxious palpation of the distal femurin sarcoma- and sham-injected animals.

The number of spontaneous flinches and time to spent guarding,representative of nociceptive behavior, were recorded simultaneouslyduring a 2-min observation period. Guarding was defined as the time thehindpaw was held aloft while ambulatory and flinches were the number oftimes the animal held the limb aloft.

Normal limb use during spontaneous ambulation was scored on a scale of 5to 0: (5) normal use, and (0) complete lack of limb use.

Forced ambulatory guarding was determined using a rotarod (ColumbusInstruments, Columbus, Ohio). The rotarod machine has a revolving rodand is equipped with speed, acceleration, and sensitivity controls. Theanimals were placed on the rod with X4 speed, 8.0 acceleration, and 2.5sensitivity. Forced ambulatory guarding was rated on a scale of 5–0: (5)normal use, and (0) complete lack of use.

After a normally non-noxious palpation of the distal femur in animalsevery second for 2 min, the animals were placed in the observation boxand their palpation-induced guarding and palpation-induced flinchingwere measured for an additional 2 min.

Treatment with anti-NGF antibody. On day 6 and day 13, sarcoma-injectedanimals were intraperitoneally (i.p.) injected with anti-NGF antibody911 at 10 mg/kg (sarc+anti-NGF, n=5), or sarcoma- and sham-injectedanimals were injected (i.p.) with saline (sham+veh or sarc+veh, n=5 foreach condition). All animals were behaviorally analyzed on days 10 and14.

Evaluation of ongoing pain behaviors. As shown in FIG. 1,sarcoma-injected animals (administered with saline) developed ongoingpain behaviors as assessed by spontaneous guarding and spontaneousflinching (both p<0.05, ANOVA) as compared to sham-injected animals(administered with saline). FIG. 1 also shows that i.p. administrationof anti-NGF antibody 911 significantly reduced spontaneous guarding andspontaneous flinching in sarcoma-injected mice on day 10 and day 14post-sarcoma implantation as compared to administration of saline tosarcoma-injected mice (p<0.05, ANOVA, for both spontaneous guarding andspontaneous flinching). These results indicate anti-NGF antibody 911reduces ongoing pain in sarcoma-injected mice.

Evaluation of ambulatory pain behaviors. As shown in FIG. 2,sarcoma-injected animals (administered with saline) developed ambulatorypain behaviors as assessed by limb use and forced ambulation guarding(rotarod) (both p<0.05, ANOVA) as compared to sham-injected animals(administered with saline). FIG. 2 also shows that i.p. administrationof anti-NGF antibody 911 significantly increased (closer to normal) limbuse score and forced ambulatory guarding score in sarcoma-injected miceon day 10 and day 14 post-sarcoma implantation as compared toadministration of saline to sarcoma-injected mice (p<0.05, ANOVA, forboth limb use and force ambulatory guarding). These results indicateanti-NGF antibody 911 reduces ambulatory pain in sarcoma-injected mice.

Evaluation of touch-evoked pain behaviors. As shown in FIG. 3,sarcoma-injected animals (administered with saline) developedtouch-evoked pain behaviors as assessed by palpation-induced guardingand palpation-induced flinching (both p<0.05, ANOVA) as compared tosham-injected animals (administered with saline). FIG. 3 also shows thati.p. administration of anti-NGF antibody 911 significantly reducedpalpation-induced guarding and palpation-induced flinching insarcoma-injected mice on day 10 and day 14 post-sarcoma implantation ascompared to administration of saline to sarcoma-injected mice (p<0.05,ANOVA, for both palpation-induced guarding and palpation-inducedflinching). These results indicate anti-NGF antibody 911 reducestouch-evoked pain in sarcoma-injected mice.

Example 2 Anti-NGF Monoclonal Antibody is Effective in Treating BoneCancer Pain and Reduces Several Neurochemical Changes Associated withPeripheral and Central Sensitization in the Dorsal Root Ganglion andSpinal Cord

Methods

Animals. Experiments were performed on a total of 158 adult male C3H/HeJmice (Jackson Laboratories, Bar Harbor, Me.), weighing 20–25 g. The micewere housed in accordance with the National Institutes of Healthguidelines under specific pathogen free (SPF) conditions in autoclavedcages maintained at 22° C. with a 12-hour alternating light and darkcycle and were given autoclaved food and water ad libitum.

Culture and injection of tumor cells. Osteolytic murine sarcoma cellswere obtained (NCTC 2472, ATCC, Rockville, Md.), stably transfected withgreen fluorescent protein (GFP) and maintained as previously describedby Sabino et al., Cancer Res. 62: 7343–9 (2002).

Injection of tumor cells were performed as previously described. Honoreet al., Nat. Med. 6: 521–8 (2000); Honore et al., Neuroscience98:585–598 (2000); Luger et al., Cancer Research 61: 4038–4047 (2001).In brief, following induction of general anesthesia with sodiumpentobarbital (50 mg/kg, i.p.), an arthrotomy was performed exposing thecondyles of the distal femur. Hank's buffered sterile saline (HBSS,Sigma Chemical Co., St. Louis, Mo.; 20 μl; sham, n=40) or mediacontaining 10⁵ osteolytic murine sarcoma cells (20 μl, NCTC 2472, ATCC,Rockville, Md; sarcoma, n=90) was injected into the intramedullary spaceof the mouse femur and the injection site sealed with dental amalgam(Dentsply, Milford, Del.), followed by irrigation with sterile filteredwater. A day 14 endpoint was used, as this is the time point when thetumor is still confined to the bone and there is maximal presentation ofcancer-related pain behaviors and maximal changes in expression ofneurochemical markers of peripheral and central sensitization. Shamanimals were used for control analysis of neurochemical changes and bonehistology, as naïve animals were not significantly differentbehaviorally, neurochemically or histologically.

Treatment with anti-NGF antibody. To assess the effect of the anti-NGFantibody treatment on pain-related behaviors, neurochemical changes,tumor growth and bone destruction, the anti-NGF antibody (mAb 911,described in Hongo, et al., Hybridoma 19:215–227 (2000)) wasadministered (10 mg/kg/every 5 days, i.p.) beginning 6 dayspost-injection when observable bone destruction began and was terminatedat 14 days post-injection, when significant bone destruction and painbehaviors were observed. The doses used in the current study caused noadverse effects, such as hypoalgesia, in naive mice. To monitor thegeneral health of the mice, weights were recorded at the beginning andend of the experiments.

Mice were randomly placed into treatment groups receiving either sterilesaline (sham+vehicle: n=28; sarcoma+vehicle: n=35; 1.4 μl/g/every 5days, i.p.) or an anti-NGF antibody (sham+anti-NGF; n=4;sarcoma+anti-NGF: n=23, 10 mg/kg/every 5 days, i.p.) weekly. Forbehavioral comparison of anti-NGF antibody to morphine sulfate, micewere given a dose of morphine 15 minutes prior to behavioral testing(naïve: n=6; sham+vehicle: n=8; sarcoma+vehicle: n=8; sarcoma+anti-NGF:n=8; sarcoma+morphine 10 mg/kg, i.p.: n=8; sarcoma+morphine 30 mg/kg,i.p.: n=8). For thermal and mechanical sensitivity testing and theassessment of hindpaw skin innervation, mice were divided into twotreatment groups receiving either sterile saline (naïve+vehicle: n=11)or an anti-NGF antibody (naïve+anti-NGF: n=11, 10 mg/kg/every 5 days,i.p.) weekly for 2 weeks.

Characterization of the anti-NGF antibody. The NGF antagonist antibody(mAb 911) is effective in blocking the binding of NGF to the Trk A andp75 NGF receptors and inhibiting Trk A autophosphorylation and blockingof NGF-dependent survival of dorsal root ganglion sensory neurons.Hongo, et al., Hybridoma 19:215–227 (2000).

Euthanasia and processing of tissue. Mice were sacrificed at day 14 posttumor injection and the tissues were processed for immunohistochemicalanalysis of spinal cord, dorsal root ganglia (DRG) as previouslydescribed and hindpaw skin. Honore et al., Nat. Med. 6: 521–8 (2000);Luger et al., Cancer Research 61: 4038–4047 (2001). Briefly, micereceived a normally non-noxious mechanical stimulation of the injectedknee 1.5 hours prior to euthanasia for induction of c-Fos expression.Honore et al., Neuroscience 98:585–598 (2000); Hunt et al., Nature 328:632–634 (1987). Following this manipulation, mice were euthanized withCO₂ and perfused intracardially with 12 ml 0.1 M phosphate bufferedsaline (PBS) followed by 25 ml 4% formaldehyde/12.5% picric acidsolution.

Spinal cord segments (L2–L4), DRG (L1–L5) and plantar skin were removed,post-fixed in the perfusion fixative and cryoprotected in 30% sucrosefor 24 hours. Serial frozen spinal cord and skin sections, 60 μm thick,were cut on a sliding microtome, collected in PBS, and processed as freefloating sections. Serial DRG sections, 15 μm thick, were cut on acryostat and thaw-mounted on gelatin-coated slides for processing.

Following sectioning, DRG, spinal cord and plantar skin sections werebriefly rinsed in PBS and then incubated in blocking solution (3% normaldonkey serum (NDS) 0.3% Triton X-100 in PBS) for 1 hr followed byincubation overnight in the primary antibody. Spinal cord sections wereimmunostained for c-Fos protein (1:2000, Oncogene Research, San Diego,Calif.) and dynorphin (polyclonal guinea pig anti-dynorphin, 1:1,000,Neuromics, Minneapolis, Minn.). DRG sections were immunostained foractivating transcription factor 3 (ATF-3) (polyclonal rabbit anti-ATF-3,1:500, Santa Cruz Biotechnologies, Santa Cruz, Calif.) and CD68 (ED-1;polyclonal rat anti-CD68, 1:5,000, Serotec, Raleigh, N.C.). Skinsections were immunostained for calcitonin gene related peptide (CGRP)(1:15,000; Sigma, St. Louis, Mo.), tyrosine hydroxylase (TOH)(polyclonal rabbit anti-TOH, 1:2,000, Chemicon, Temecula, Calif.) andneurofilament H (Clone RT97) (polyclonal rabbit anti-RT-97, 1:2,500,Chemicon, Temecula, Calif.).

After incubation in primary antibody, sections were rinsed in PBS andthen incubated in the secondary antibody solution for 3 hr. Secondaryantibodies, conjugated to Cy3 or biotin (Jackson ImmunoResearch, WestGrove, Pa.), were used at 1:600 or 1:500 respectively. To detectsecondary antibodies conjugated to biotin: following secondaryincubation, sections were rinsed in PBS and incubated in Cy3 conjugatedstreptavidin (1:4000; Jackson ImmunoResearch) for 45 minutes. To confirmspecificity of the primary antibodies, controls included omission of theprimary antibody or preabsorption with the corresponding syntheticpeptide. Following immunostaining procedures, spinal cord, and plantarskin sections were mounted onto gelatin-coated slides. Mounted sectionsof skin, spinal cord and DRG were then dehydrated in alcohol gradients(70, 90, 100%), cleared in xylene and coverslips were mounted with DPX(Fluka, Switzerland).

Following radiological examination, at day 14, right (internal control)and left (tumor-bearing) femora were fixed in picric acid and 4%formalin at 4° C. overnight and decalcified in 10% EDTA (Sigma., St.Louis, Mo.) for no more than 14 days. Bones were then embedded inparaffin. Femoral sections, 5 μm thick were cut in the lateral plane andstained with tartrate-resistant acid phosphatase (TRAP) and hematoxylinand eosin (H&E) to visualize histological features of the normal bonemarrow, tumor, osteoclasts and macrophages. To visualize sarcoma cellsusing fluorescence microscopy, femoral sections 5 μm thick were stainedwith an antibody raised against green fluorescent protein (GFP) (rabbitanti-GFP, 1:6,000, Molecular Probes, Eugene, Oreg.). GFP staining wasperformed using TSA-Plus Cyanine 3 System (PerkinElmer Life Sciences,Inc., Boston, Mass.), as previously described by Sevcik et al., Pain111: 169–80 (2004).

Immunohistochemical analysis of the sham and cancerous femora wasperformed on decalcified, paraffin embedded 14 μm serial sections. TheTyramine Signal Amplification (TSA) System (Perkin Elmer life Sciences,Boston, Mass.) was used to amplify Cy3 labeled antibodies. Endogenousperoxidases were quenched by incubating the sections in 2% hydrogenperoxide for 1 hour. Sections were then rinsed three times with PBS for10 minutes and blocked in TSA blocking buffer for 1 hour. Primaryantiserum was added upon removal of the blocking buffer and allowed toincubate at room temperature overnight. Primary afferent unmyelinatedand thinly myelinated sensory nerve fibers were labeled using anantibody raised against polyclonal rabbit anti-calcitonin gene relatedpeptide (CGRP) (1:15,000; Sigma). Sections were rinsed three times inTSA wash buffer for 10 minutes followed by 45 minute incubation instreptavidin HRP (1:4,000). Sections were then rinsed three times withTSA wash buffer for 10 minutes. CY3-conjugated tyramine (1:600) wasapplied to the femora for 7 minutes, washed twice with TSA wash bufferand once with PBS. Finally, the sections were air dried, dehydratedthrough an alcohol gradient (70, 90 and 100%), cleared in xylene andmounted with DPX (Fluka).

Radiographical and osteoclast and macrophage proliferation analysis ofbone. Radiographs (Faxitron X-ray Corp., Wheeling, Ill.) of dissectedfemora were obtained at the day 14 time point to optimally assess bonedestruction. Images were captured on Kodak Min-R 2000 mammography film(Eastman Kodak Co., Rochester, N.Y.; exposure settings: 7 sec, 21 kVp).The extent of tumor-induced femoral bone destruction was radiologicallyassessed in the lateral plane of whole bone images at 5× magnificationusing a 0 to 5 scale (0, normal bone with no signs of destruction and 5,full-thickness bicortical bone loss). Honore et al., Nat. Med. 6: 521–8(2000); Honore et al., Neuroscience 98:585–598 (2000); Luger et al.,Cancer Research 61: 4038–4047 (2001).

Osteoclast and tumor associated macrophage (TAMs) proliferation weredetermined by quantifying the number of TRAP+ osteoclasts or TAMs onTRAP stained femoral sections as previously described. Honore et al.,Nat. Med. 6: 521–8 (2000); Honore et al., Neuroscience 98:585–598(2000). Briefly, TAMs are differentiated histologically from osteoclastson femoral sections stained with TRAP as TRAP+ cells that are freely andmultidimensionally dispersed throughout the tumor mass. Macrophageswithin the bone become activated due to tumor released factors thatstimulate the cells, and the cellular appearance of these activated TAMsis marked by their highly irregular surface, multiple lamellipodia andphagocytic vacuoles. Osteoclasts are histologically differentiated ascells appearing TRAP+ and which are closely associated with regions ofbone resorption. These cells are multinucleate and are found along thecortical and trabecular bone. Results are expressed as the mean numberof osteoclasts per mm or TAMs per mm², respectively.

Quantification of tumor growth. Femora containing GFP-expressing sarcomacells were imaged using a yellow 515 nm long pass emission filter on aNikon E600 fluorescence microscope equipped with a SPOT II digitalcamera utilizing SPOT image capture software (Diagnostic Instruments,Sterling Heights, Mich.). The total area of intramedullary space and thepercent of intramedullary space occupied by tumor were calculated usingImage Pro Plus v3.0 software (Media Cybernetics, Silver Spring, Md.).Sabino et al., Cancer Res. 62: 7343–9 (2002); Sevcik et al., Pain 111:169–80 (2004). The tumor characteristics of sarcoma cells transfectedwith GFP, such as growth rates, rate of bone resorption and the abilityto induce bone cancer-related pain behaviors, were temporally,behaviorally and physically identical to non-transfected sarcoma cells.Sabino et al., Cancer Res. 62: 7343–9 (2002)

Quantification of sensory fibers in bone. The number of sensory nervefibers was determined as previously described. Mach et al., Neuroscience113:155–66 (2002). Briefly, the number of CGRP positive fibers in threebone regions (proximal, distal and diaphyseal) and the three bonetissues (periosteum, mineralized bone and marrow) were quantified. Onlynerve fibers greater than 30 μm in length were included in the analysis.Six sections per animal were analyzed, and the fibers counted wereexpressed as fibers per total bone area.

Quantification of spinal cord, dorsal root ganglion and hindpaw skin.Fluorescently labeled spinal cord, DRG and skin tissue sections wereanalyzed using either an MRC 1024 confocal microscope imaging system(Bio-Rad, Philadelphia, Pa.), or a SPOT II digital camera on an OlympusBX-60 fluorescence microscope with SPOT image capture software(Diagnostic Instruments, Inc.).

The number of DRG neurons expressing activating transcription factor 3(ATF-3) were counted under 200× magnification with a 1 cm² eyepiecegrid. The total number of neurons (small, medium and large) wasdetermined by counting both labeled and non-labeled neuronal cellsbodies (non-labeled cell bodies exhibit background labeling that couldbe examined through a rhodamine or FITC filter) and results expressed aspercent of total number of neurons which express ATF-3-immunoreactivity(IR). To prevent duplicate counting of neuronal cell bodies, counts wereconducted on every fourth serial section for each marker. To quantifythe activated or infiltrating macrophages in DRG, SPOT camera grey scaleimages were obtained on a minimum of four ipsilateral and contralateralDRG sections per animal and analyzed using Image Pro Plus version 3.0software (Media Cybernetics). For each image, regions of the DRGcontaining only sensory neuronal cell bodies (excluding peripheralnerve) were outlined. While viewing the monitor, upper and lowerthresholds of gray level density were set such that only specificCD68-IR cellular profiles were discriminated from the background in theoutlined DRG. The number of cellular profiles was counted per sectionautomatically. The SPOT camera output had been calibrated in Image ProPlus such that the actual area of each outlined region within acquiredimages could be determined. The section values for CD68-IR cellularprofiles and outlined areas were summed for each animal and results wereexpressed as total number of CD68-IR cellular profiles per unit area(mm²).

Quantification was carried out in spinal cord sections at lumbar levelsL2–L4 as these spinal segments receive significant afferent input fromthe L1–L3 DRGs, which are the principal ganglia that provide afferentinput to the mouse femur. Edoff et al., Cell & Tissue Research 299:193–200 (2000); Molander C, J. Comp. Neurol. 260: 246–255 (1987);Puigdellivol-Sanchez A et al., the Anatomical Record 260: 180–188(2000); Puigdellivol-Sanchez A et al., Neurosci. Lett. 251: 169–172(1998). Quantification of spinal cord sections for dynorphin wasobtained from 4 randomly selected L2–L4 coronal spinal cord sections peranimal. The number of dynorphin-IR neurons in spinal cord laminae III–VIwere counted at 100× magnification and expressed as mean number ofneurons per 60 μm L2–L4 section per animal. The number of c-Fos-IRneurons was counted in laminae III–VI of the dorsal horn in 8 randomlyselected L3/L4 coronal spinal cord sections per animal. To be consideredc-Fos-IR, the immunofluorescence threshold of the nuclear profile wasset at three times the mean background immunofluorescence level of thetissue section. Results are given as mean number of c-Fos-IR neurons perspinal cord section.

Quantification of epidermal innervation density was performed on 4randomly selected plantar hindpaw skin sections per animal. The totalnumber of CGRP, TOH and RT97-IR nerve fibers were counted at 200×magnification. Counting rules were established to count only singleintra-epidermal fibers and not multiple branches of the same fiber.McCarthy et al., Neurology 45: 1848–55 (1995). The total length ofepidermis in all sections quantified was measured using a 1 cm² eyepiecegrid. Only nerve fibers that were at least 25 μm in length, andprojected into the superficial epidermis were counted. Results are givenas the mean number of intra-epidermal nerve fibers per mm length peranimal.

Behavioral analysis. Mice were tested for bone cancer pain-relatedbehaviors 10 and 14 days following sham or tumor injections when painbehaviors are significantly evident to assess the efficacy of anti-NGFtreatment. The anti-NGF treatment was compared to morphine (Baxter,Deerfield, Ill.; 10 mg/kg, i.p.) treatment and was administered 15minutes prior to behavioral testing to ensure that animals were testedwithin the therapeutic window of drug action. Hasselstrom et al.,Pharmacology & Toxicology 79: 40–6 (1996).

Mice were also tested 8, 10, 12 and 14 days following tumor or shaminjections to assess efficacy of anti-NGF treatment (10 mg/kg/every 5days, i.p.) in attenuating pain-related behaviors throughout theprogression of the disease. Animals were observed over a 2-minute periodand ongoing and palpation-evoked bone cancer pain behaviors wereanalyzed, as previously described. Luger et al., Pain 99:397–406 (2002);Sabino et al., Cancer Res. 62: 7343–9 (2002); Sabino et al.,International Journal of Cancer 104: 550–558 (2003). Briefly, the numberof hindpaw flinches and time spent guarding were recorded as measures ofongoing pain, as these measures mirror patients in a clinical settingwith bone cancer who protect or suspend their tumor-bearing limb. In ourmodel, movement-evoked pain due to palpation of the injected limb wasevaluated using previously validated tests. Luger et al., CancerResearch 61: 4038–4047 (2001); Sabino et al., International J. of Cancer104: 550–558 (2003); Sevcik et al., Pain 111: 169–80 (2004).Palpation-evoked pain behaviors were examined where animals received anormally non-noxious palpation to the tumor- or sham-injected limb fortwo minutes prior to observation. Luger et al., Cancer Research 61:4038–4047 (2001); Sevcik et al., Pain 111: 169–80 (2004). Mice weremonitored over a 2-minute period, and the number of flinches and timespent guarding were recorded. Palpation-evoked behavior tests weredeveloped to reflect the clinical condition when patients with bonecancer experience pain following normally non-noxious movement of thetumor-bearing limb.

Following a 15 minute acclimation period, thermal and mechanicalsensitivity were measured in naïve and naïve+anti-NGF animals to assesswhether the normal pain threshold responses were altered with anti-NGFtreatment. Thermal sensitivity was measured using a Thermal PawStimulator (University of California, San Diego, San Diego, Calif.). Theintensity of radiant heat was adjusted so that the naïve animalsresponded to the heat by elevating the hindpaw approximately nineseconds after the heat was initiated. Choi et al., Life Sci. 73: 471–85(2003). The mice were allowed 5 minutes to recover between each trial. Asingle trial consisted of 4 measurements per hindpaw, the longestlatency was dropped and the remaining 3 measurements were averaged.Mechanical sensitivity was measured using a previously validated method.Chaplan et al., J. Neuroscience Methods 53: 55–63 (1994) Von Freyfilaments (Stoelting Co., Wood Dale, Ill.) were applied to the hindpawof the animals, and the withdrawal threshold was determined byincreasing and decreasing the stimulus intensity between 0.2 and 15.1gram equivalents of force. A positive response was noted if the paw wasquickly withdrawn.

RTPCR Analysis of mRNA levels of NGF in the 2472 cell line. Total RNAfrom triplicate mouse tissue samples or 2472 sarcoma cells was preparedaccording to manufacturer's instructions using the RNeasy micro kit(Qiagen), and the RNA was quantified using Ribogreen reagent (MolecularProbes). Two-step RT-PCR was performed using the TaqMan Gold RT-PCR kit(Applied Biosystems). The RNA was reverse transcribed using randomhexamers, and the cDNA was amplified using a primer/probe set specificfor NGF (muNGF-187F: GGGCTGGATGGCATGCT (SEQ ID NO:3), muNGF-256R:GCGTCCTTGGCAAAACCTT (SEQ ID NO:4), muNGF-208T: CCAAGCTCACCTCAGTGTCTGGGCC(SEQ ID NO:5)). The samples were analyzed in duplicate from the RT leveland normalized to total RNA input.

Statistical analysis. The SPSS version 11 computer statistics package(SPSS, Chicago, Ill.) was used to perform statistical analyses. Mixedeffects linear regression modeling was used to analyze the repeatedmeasures data, which can accommodate subjects measured at differing timeintervals, include both fixed and time-varying covariates, and canestimate individual rates of change. Each dependent outcome variable wascompared across groups using a non-parametric analysis of variance(Kruskal-Wallis). Significant Kruskal-Wallis analyses were followed bynon-parametric post-hoc comparisons between pairs of groups using theMann-Whitney U test. Results were considered statistically significantat P<0.05. In all cases, the investigator was blind to the experimentalstatus of each animal.

Results

Anti-NGF administration had no effect on disease progression ormacrophage infiltration in the bone. The effects of anti-NGF treatmenton bone destruction, osteoclast proliferation and tumor growth wereexamined at day 14 post tumor injection. Sham-injected mice did notdemonstrate significant bone destruction (bone score 0.9±0.4; FIG. 4 a),osteoclast proliferation (4.6±0.4 osteoclasts/mm) or tumor growth (FIG.4 d), as assessed by radiological, TRAP and H&E/GFP analysis,respectively as compared to sarcoma-injected mice. In sarcoma+vehiclemice, there was extensive bone destruction as observed and characterizedby multifocal radiolucencies (bone score 3.5±0.2; FIG. 4 b), markedincrease in the number of osteoclasts (4.0±0.7 osteoclasts/mm) and thetumor had completely filled the intramedullary space (100±0.0% ofintramedullary space; FIG. 4 e). Treatment of tumor-bearing mice withanti-NGF from day 6 to day 14 post tumor injection resulted in nosignificant change in bone resorption (3.1±0.6; FIG. 4 c), no reductionin sarcoma-induced osteoclast proliferation (3.5±0.1 osteoclasts/mm) ortumor growth (98.0±0.9% of intramedullary space; FIG. 4 f) as comparedto sarcoma+vehicle animals.

Fourteen days following tumor injection, sarcoma+vehicle mice displayedan up regulation of TAMs (39.8±12.6 TAMs/mm² ) as compared tosham+vehicle control mice (0.0±0.0 TAMs/mm²). Anti-NGF treatment ofsarcoma-injected mice (29.5±7.3 TAMs/mm²) did not significantly alterthis TAM infiltration, as seen in the sarcoma+vehicle mice.

Anti-NGF treatment had no observable effect on sensory or sympatheticinnervation in bone or skin. Thinly myelinated or unmyelinatedpeptidergic sensory nerve fibers were labeled with an antibody raisedagainst calcitonin gene related peptide (CGRP). CGRP-IR nerve fiberswere found throughout the entire bone (periosteum, mineralized bone andbone marrow) of both naïve+vehicle (12.2±0.3 fibers/mm) andnaïve+anti-NGF (13.0±0.8 fibers/mm) animals.

Thinly myelinated or unmyelinated peptidergic sensory nerve fibers(CGRP-IR), large myelinated sensory fibers (RT97-IR) and noradrenergicsympathetic nerve fibers (TOH-IR) were analyzed in the hindpaw plantarskin by antibodies raised against CGRP, RT-97 and TOH, respectively.There was no significant difference between the intensity or density ofCGRP positive fibers in sarcoma+vehicle (12.0±0.8 fibers/mm) andsarcoma+anti-NGF (12.5±0.6 fibers/mm) hindpaw skin samples (FIGS. 5 aand 5 b). Similarly, there was no difference in intensity or density ofCGRP positive fibers between naïve+vehicle (FIG. 5 c, n=8) mice andnaïve+anti-NGF (FIG. 5 d, n=8) mice. There was no change in the numberof nerve fibers expressing CGRP in sarcoma-injected and naïve mice (a,bvs. c,d). Differences in the density and intensity of RT97 positive andTOH positive fibers were also undetectable in sarcoma+vehicle (7.3±0.7RT97+ fibers/mm; 3.1±0.7 TOH+ fibers/mm) and sarcoma+anti-NGF treated(7.3±0.7 RT97+ fibers/mm; 3.6±0.7 TOH+ fibers/mm) animals. Similarly,there was no significant difference between the intensity or density ofCGRP positive fibers in naïve+vehicle (12.5±0.5 fibers/mm) andnaïve+anti-NGF (11.9±0.7 fibers/mm) hindpaw skin samples (FIGS. 5 c and5 d). Differences in the density and intensity of RT97 positive and TOHpositive fibers were also undetectable in naïve+vehicle (10.4±0.4 RT97+fibers/mm; 3.4±0.4 TOH+ fibers/mm) and naïve+anti-NGF treated (11.9±0.7RT97+ fibers/mm; 3.0±0.8 TOH+ fibers/mm) animals. There were nosignificant observable differences between the intensity or density ofCGRP, RT97 or TOH positive fibers in the skin samples of sarcoma+vehicleand sarcoma+anti-NGF versus the naïve+vehicle and naïve+anti-NGFanimals.

Anti-NGF antibody therapy significantly reduced bone cancer painbehaviors. Sarcoma+vehicle mice demonstrated a greater time spentguarding as compared to the sham+vehicle controls (FIG. 6 a).Additionally, sarcoma+vehicle mice exhibited an increased number offlinches as compared to sham+vehicle controls (FIG. 6 b). Administrationof anti-NGF (from day 6 to day 14) in sarcoma-injected micesignificantly attenuated spontaneous guarding as compared tosarcoma+vehicle mice (FIG. 6 a). Anti-NGF treatment also significantlyreduced spontaneous flinching in sarcoma-injected mice (FIG. 6 b).

Movement-evoked pain was analyzed by measuring palpation-inducedresponses. Sarcoma+vehicle mice demonstrated a greater time spentguarding after palpation as compared to the sham+vehicle controls (FIG.6 c). Sarcoma+vehicle mice also exhibited an increased number offlinches after palpation as compared to sham+vehicle controls (FIG. 6d). Anti-NGF treatment in sarcoma-injected mice significantly reducedboth palpation-evoked guarding (FIG. 6 c) and palpation-evoked flinching(FIG. 6 d). In preliminary studies, no significant behavioraldifferences or side effects were observed between sham-operated animalsreceiving either vehicle or anti-NGF.

FIG. 6 shows that anti-NGF treatment (n=8) from 6 to 14 days post tumorinjection (triangle) significantly reduced ongoing and palpation-evokedpain behaviors on days 10, 12 and 14 as compared to sarcoma+vehicle(n=8) (square), and was significantly reduced to sham levels at day 10for all parameters (diamond). At all time points, sham+vehicle (n=8) aresignificantly different from sarcoma+vehicle. Thus, anti-NGF treatment(10 mg/kg, i.p., every 5 days) attenuated both ongoing andmovement-evoked bone cancer pain behaviors throughout the progression ofthe disease.

Anti-NGF treatment had no effect on baseline thermal or mechanicalthresholds and was comparable to the efficacy of morphine in reducingbone cancer pain. There was no significant increase in latency of pawwithdrawal to a thermal stimulus or increase in threshold of mechanicalstimulation with anti-NGF administration as compared to normal painthresholds. Anti-NGF treatment had no effect on either normal thermalresponse (FIG. 7 a) as compared to naïve+vehicle or normal mechanicalstimulation (FIG. 7 b) as compared to naïve+vehicle.

Animals were tested to compare the efficacy of morphine sulfate (MS) tothe anti-NGF antibody in reducing bone cancer-related behaviors.Behavioral assessment on days 10 and 14 revealed that sarcoma+vehicleanimals showed statistically longer time guarding (FIG. 7 c) andincreased time guarding in response to palpation (FIG. 7 d) of theinjected limb as compared to sham+vehicle animals. Treatment with eitheranti-NGF (10 mg/kg/every 5 days, i.p.) or morphine sulfate (10 mg/kg, or30 mg/kg i.p.) significantly reduced both ongoing and movement-evokedguarding behaviors at days 10 and 14 post tumor injection (FIG. 7 c, 7d), as compared to sarcoma+vehicle mice. Anti-NGF treatmentsignificantly attenuated the bone cancer-related pain behaviors moreeffectively as compared to morphine doses of either 10 mg/kg or 30 mg/kg(P<0.05 vs. Sarcoma+anti-NGF).

Anti-NGF treatment modulated peripheral changes induced by bone cancerin the DRG. Activating transcription factor-3 (ATF-3), which is in theATF/CREB family, has previously been shown to be up-regulated in a modelof peripheral nerve injury. Tsujino et al., Molecular & CellularNeurosciences 15:170–82 (2000). This up-regulation is seen in sensoryand motor neuron cell bodies and is known to label injured neurons.There was significant increase in the percentage of ATF-3-IR neurons inL2 DRG ipsilateral to the sarcoma-injected femur (14.0±5.9% of totalneurons in L2 expressed ATF-3; FIG. 8 a) as compared to sham+vehicle(1.6±0.5% of total neurons in L2 expressed ATF-3). Treatment withanti-NGF significantly attenuated the expression of ATF-3 (2.6±1.0% oftotal neurons in L2 expressed ATF-3; FIG. 8 b) 14 days post tumorinjection.

Macrophage infiltration has been shown to be up regulated due toperipheral nerve damage. Abbadie et al., Proc. Natl. Acad. Sci. USA.100: 7947–52 (2003); Myers et al., Exp. Neurol. 141: 94–101 (1996);Tofaris et al., J. Neurosci. 22: 6696–703 (2002). An antibody raisedagainst CD68 (ED-1), a lysosomal protein expressed by activated tissuemacrophages, was used to assess macrophage infiltration insarcoma-injected mice. There was an up regulation in the number ofCD68-IR neurons in the ipsilateral DRG of sarcoma+vehicle mice(119.6±12.1 cellular profiles/L2 ipsilateral DRG; FIG. 8 c) compared tosham+vehicle (80.6±6.0 cellular profiles/L2 ipsilateral DRG). Anti-NGFtreatment significantly reduced the up-regulation of CD68-IR neurons inthe ipsilateral DRG (92.0±9.9 cellular profiles/L2 ipsilateral DRG; FIG.8 d) in sarcoma-injected mice, indicating a significant reduction in thenumber of activated and inflitrating microphage within the ipsilateralL2 DRG of tumor bearing animals.

Anti-NGF treatment modulated central changes induced by bone cancer inthe spinal cord. Expression of dynorphin has been shown to be involvedin the maintenance of chronic pain. Vanderah et al., Pain 92: 5–9(2001). Dynorphin expression has also been shown to be up-regulated inthe dorsal horn of the spinal cord in several persistent pain states.Iadarola, et al., Brain Res. 455: 205–212 (1988); Noguchi et al.,Molecular Brain Research 10: 227–233 (1991); Schwei et al., J. Neurosci.19: 10886–97 (1999). In sham+vehicle mice, a small amount of spinalneurons expressed dynorphin in deep spinal laminae (2.3±1.1 dyn-IRneurons/L3/L4 section). In contrast, sarcoma+vehicle mice expressedsignificantly more dynorphin-IR neurons (6.0±0.5 dyn-IR neurons/L3/L4section; FIG. 9A). Anti-NGF treatment significantly reduced theup-regulation of dynorphin expression (2.0±0.6 dyn-IR neurons/L3/L4section; FIG. 9B) in sarcoma-injected mice.

Immediate-early gene activation was prevented by anti-NGF treatment. Theexpression of c-Fos in the deep dorsal horn (laminae III–VI) has beenutilized as a marker of central sensitization in sarcoma-induced bonecancer pain states. Honore et al., Nat. Med. 6: 521–8 (2000); Honore etal., Neuroscience 98:585–598 (2000); Luger et al., Cancer Research 61:4038–4047 (2001); Schwei et al., J. Neurosci. 19: 10886–97 (1999).Normal, non-noxious palpation of sham-operated animals resulted inminimal expression of c-Fos in deep laminae. Sabino et al., Cancer Res.62: 7343–9 (2002). In the bone cancer state, sarcoma+vehicle miceexhibited an increased number of c-Fos-IR neurons (27.7±4.9; cFos-IRneurons/L3/L4 section; FIG. 9C) and treatment with anti-NGFsignificantly reduced this expression (11.1±1.9; cFos-IR neurons/L3/L4section; FIG. 9D).

RT PCR Results. In order to see whether the sarcoma tumor cells were apossible source of NGF, 2472 cells grown in culture were assessed fortheir level of NGF mRNA by RT-PCR. These levels were compared to severalnormal tissues of the mouse, as well as the level of NGF mRNA from themale mouse salivary gland, a source of aberrantly high exocrine NGF. Asseen in Table 3 below, sarcoma 2472 cells in vitro contained readilydetectable NGF mRNA. This level is in the range of NGF mRNA levelsobtained from normal tissues expressing high levels of NGF mRNA, such asiris. Shelton et al., Proc. Natl. Acad. Sci. USA. 81:7951–5 (1984).However, this level is several orders of magnitude below the level ofNGF mRNA present in male mouse salivary gland.

TABLE 3 RT PCR data showing level of NGF expression Tissue typeArbitrary Units Brain 1.2 ± 0.8 Atria 1.9 ± 0.7 2472 cells   8 ± 1.1Iris 8.8 ± 3.6 Submaxillary Gland 1359.1 ± 583.7 

Example 3 Effect of Anti-NGF Monoclonal Antibody in Treating Bone CancerPain in a Murine Model Developed by Intramedullary Injection ofOsteoblastic Prostate Tumor Cells into the Femur

Methods

Murine prostate model of bone cancer pain. A murine prostate model ofbone cancer pain was used to assess the efficacy of treatment withanti-NGF antibody 911 (a mouse monoclonal antibody; see Hongo, et al.,Hybridoma 19:215–227 (2000)). Osteoblastic canine carcinoma (ACE-1, giftfrom Dr. Thomas J. Rosol, Ohio State Univeristy) cells were maintainedand injections of tumor cells were performed as previously described.Sabino et al., Cancer Res. 62: 7343–7349, 2002; Honore et al., NatureMedicine 6: 521–528, 2000; Honore et al., Prog. Brain Res. 129: 389–397,2000; Luger et al., Cancer Research 61: 4038–4047, 2001. In brief, ACE-1cells were grown in media at 37° C. and 5% CO₂. The cells are grown inT75 flasks (7.5 cm²) and passaged at 80–90% confluency, twice per week.Only passages between 3 and 11 were used in this study. On day 0,following induction of general anesthesia with sodium pentobarbital (50mg/kg, i.p.), an arthrotomy was performed exposing the condyles of thedistal femur. Hank's buffered sterile saline (HBSS, Sigma Chemical Co.,St. Louis, Mo.; 20 μl; sham, n=7) or media containing 10⁵ osteoblasticcanine ACE-1 cells (20 μl, ACE-1, n=60) was injected into theintramedullary space of the mouse femur and the injection site sealedwith dental amalgam (Dentsply, Milford, Del.), followed by irrigationwith sterile filtered water. Experiments were performed on a total of 898–10 week old adult male athymic nude mice (Harlan Laboratories,Madison, Wis.), weighing 20–32 g. The mice were housed in accordancewith the National Institutes of Health guidelines under specificpathogen free (SPF) conditions in autoclaved cages maintained at 22° C.with a 12-hour alternating light and dark cycle and were givenautoclaved food and water ad libitum.

A day 19 post-injection endpoint was used, as this is the time pointwhen the tumor is still confined to the bone, there is maximalpresentation of cancer-related pain behaviors and tumor-induced boneremodeling. Sham animals were used for control analysis of behavioralexperiments and bone histology/immunohistochemistry, as naïve animalswere not significantly different from sham behaviorally 9 dayspost-tumor injection.

Treatment with anti-NGF antibody or morphine. On days 7, 12, and 17 posttumor-injection, ACE-1-injected animals were intraperitoneally (i.p.)injected with anti-NGF antibody 911 at 10 mg/kg (ACE-1+anti-NGF, n=9);ACE-1-injected animals were injected (i.p.) with saline (ACE-1+veh,n=21; 1.4 μl/kg); and sham-injected animals were injected (i.p.) withsaline (sham+veh, n=7). All animals were behaviorally analyzed betweendays 7 and 19.

For behavioral comparison of anti-NGF antibody to morphine sulfate, micewere given an acute dose of morphine 15 minutes prior to behavioraltesting (naïve: n=6; sham: n=7; ACE-1+vehicle: n=7; ACE-1+anti-NGF: n=7;ACE-1+morphine 10 mg/kg, s.c.: n=8; ACE-1+morphine 30 mg/kg, s.c.: n=8).For thermal and mechanical sensitivity testing and the assessment ofhindpaw skin innervation, naïve mice were divided into two treatmentgroups receiving either sterile saline (naïve+vehicle: n=8) or anti-NGFantibody (naïve+anti-NGF: n=8, 10 mg/kg, i.p.).

Behavioral analysis. Animals were tested for pain-related behaviors bothprior to and on day 7, 9, 11, 13, 15, 17 and 19 post-tumor implantationor sham injection. Animals were behaviorally tested using the followingtests: ongoing pain (spontaneous guarding and flinching) andmovement-evoked pain (palpation-evoked guarding and palpation-evokedflinching). Animals were placed in a clear plastic observation box witha wire mesh floor and allowed to habituate for a period of 30 min. Afteracclimation, spontaneous guarding and spontaneous flinching, wereassessed. Palpation-induced guarding and flinching were measured afterthe 2 min period of normally non-noxious palpation of the distal femurin ACE-1 and sham-injected animals. These tests were performed asdescribed in Examples 1 and 2.

Euthanasia and processing of tissue. Mice were sacrificed 19 dayspost-tumor injection and the tissues were processed forimmunohistochemical analysis of femora and hindpaw skin as previouslydescribed. Honore et al., Prog. Brain Res. 129:389–397, 2000; Honore etal., Nat. Med. 6: 521–8 (2000); Luger et al., Cancer Research 61:4038–4047 (2001). Mice were euthanized with CO₂ and perfusedintracardially with 12 ml 0.1 M phosphate buffered saline (PBS) followedby 25 ml 4% formaldehyde/12.5% picric acid solution.

Hindpaw plantar skin was removed, post-fixed in the perfusion fixative,and cryoprotected in 30% sucrose for 24 hours. Serial skin sections, 60μm thick, were cut on a sliding microtome, collected in PBS, andprocessed as free floating sections. Following sectioning, plantar skinsections were briefly rinsed in PBS and then incubated in blockingsolution (3% normal donkey serum (NDS) 0.3% Triton X-100 in PBS) for 1hr followed by incubation overnight in the primary antibody. Skinsections were immunostained for calcitonin gene related peptide (CGRP)(1:15,000; Sigma, St. Louis, Mo.), tyrosine hydroxylase (TOH)(polyclonal rabbit anti-TOH, 1:2,000, Chemicon, Temecula, Calif.) andneurofilament H (Clone RT97) (polyclonal rabbit anti-RT-97, 1:2,500,Chemicon, Temecula, Calif.).

After incubation in primary antibody, sections were rinsed in PBS andthen incubated in the secondary antibody solution for 3 hr. Secondaryantibodies, conjugated to Cy3 or biotin (Jackson ImmunoResearch, WestGrove, Pa.), were used at 1:600 or 1:500 respectively. To detectsecondary antibodies conjugated to biotin: sections were rinsed in PBSand incubated in Cy3 conjugated streptavidin (1:4000; JacksonImmunoResearch) for 45 minutes. To confirm specificity of the primaryantibodies, controls included omission of the primary antibody orpreabsorption with the corresponding synthetic peptide. Followingimmunostaining procedures, plantar skin sections were rinsed, mountedonto gelatin-coated slides. Mounted sections were then dehydrated inalcohol gradients (70, 90, 100%), cleared in xylene and coverslips weremounted with DPX (Fluka, Buchs, Switzerland).

Following radiological examination, at day 19 post-tumor injection,right (internal control) and left (tumor-bearing) femora were fixed inpicric acid and 4% formalin at 4° C. overnight and decalcified in 10%EDTA (Sigma) for no more than 14 days. Bones were then embedded inparaffin. Femoral sections, 5 μm thick were cut in the lateral plane andstained with tartrate-resistant acid phosphatase (TRAP) and hematoxylinand eosin (H&E) to visualize histological features of the normal bonemarrow, tumor, osteoclasts, osteoblasts, and macrophages (Ms).

Immunohistochemical analysis of the sham and cancerous femora wasperformed on decalcified, paraffin embedded 14 μm serial sections.Endogenous peroxidases were quenched by incubating the sections in 2%hydrogen peroxide for 1 hour. Sections were then rinsed three times withPBS for 10 minutes and blocked in TSA blocking buffer (TSA-Plus Cyanine3 System, PerkinElmer Life Sciences, Inc., Boston, Mass.) for 1 hour.Primary antiserum was added upon removal of the blocking buffer andallowed to incubate at room temperature overnight. Primary afferentunmyelinated and thinly myelinated sensory nerve fibers were labeledusing an antibody raised against polyclonal rabbit anti-calcitonin generelated peptide (CGRP) (1:15,000; Sigma). Sections were rinsed threetimes in TSA wash buffer for 10 minutes followed by 45 minute incubationin streptavidin HRP (1:4,000). Sections were then rinsed three timeswith TSA wash buffer for 10 minutes. CY3-conjugated tyramine (1:600)from the TSA-Plus Cyanine 3 System was applied to the femora for 7minutes, rinsed twice with TSA wash buffer and once with PBS. Finally,the sections were air dried, dehydrated through an alcohol gradient (70,90 and 100%), cleared in xylene and mounted with DPX (Fluka).

Radiographical analysis of bone. Radiographs (Faxitron X-ray Corp.,Wheeling, Ill.) of dissected femora were obtained at the day 19 timepoint to assess the extent of bone formation and destruction. Imageswere captured on Kodak Min-R 2000 mammography film (Eastman Kodak Co.,Rochester, N.Y.; exposure settings: 7 sec, 21 kVp). Analysis of bonedensity was used to assess the extent of tumor-induced bone remodelingradiographically in the lateral plane of whole bone images at 5×magnification. Tumor and non-tumor bearing femora (n=8 fornaïve+vehicle, sham+vehicle, ACE-1+vehicle, and ACE-1+anti-NGF) wereanalyzed using ‘ImageJ (Research Services Branch, National Institute ofMental Health, Bethesda, Md.)’ in a similar manner to a previouslydescribed protocol. Corey et al., Prostate 52: 20–33, 2002. Briefly,blank radiograph films and a standard step tablet (Eastman Kodak Co.)were used to develop a calibration curve. ImageJ was used to measureoptical density and subsequently converted to transmission as follows:transmission=1/(antilog₁₀[Optical density]). Given data are determinedfrom a negative image, thus transmission is a direct representation ofbone density. An HP ScanJet 7400c scanner used to capture sub-saturationfemoral radiographs and readings were recorded in duplicate from eachfemur. Results are presented as normalized transmission mean±SE.

Histological analysis of osteoblasts, osteoclasts, and macrophages,tumor growth and bone remodeling. Osteoblast proliferation was analyzedby quantifying the number of osteoblasts immediately in contact withregions of both tumor-induced new bone formation contained within thefemur and cortical bone throughout the entire diaphyseal intramedullaryspace for naïve animals, sham-injected, and tumor-bearing mice.Diaphyseal intramedullary space was defined as extending from theproximal distal trabeculae to the distal proximal trabeculae and wasselected for quantification as the predominant active bone remodelingoccurs in this region. Osteoblasts were identified as those cells indirect contact with the newly advancing bone matrix and arranged intypical cubiodal or columnar epithelial layer and connected to oneanother via a thin process identifiable at high magnification (200× orgreater). Results are presented as the number of osteoblasts/mm² ofdiaphyseal intramedullary space for naïve, sham-injected, andtumor-bearing mice. Osteoclast proliferation was determined byquantifying the number of TRAP+ osteoclasts at the bone/tumor interfaceand at the normal marrow/bone interface for naïve, sham-injected, andACE-1-injected mice on TRAP stained femoral sections as previouslydescribed. Honore et al., Nat. Med. 6: 521–528 (2000). In brief,osteoclasts are histologically differentiated cells appearing as TRAP+and which are closely associated with regions of bone resorption. Thesecells are multinucleate and are found in Howship's lacunae along thecortical and trabecular bone. Fawcett, D. W.; A Textbook of Histology.In: D. Dreibelbis (ed.), Bone, 11 edition, pp. 211–213. Philadelphia,Pa.: W.B. Saunders Company, 1986. Macrophage (Ms) proliferation wasdetermined by quantifying the number of TRAP+ cells that were dispersedthroughout the tumor and normal marrow not associated with the endostealsurface of the mineralized bone. Macrophages within the bone becomeactivated due to tumor released factors that stimulate the cells, andthe cellular appearance of these activated Ms is marked by their highlyirregular surface, multiple lamellipodia and phagocytic vacuoles.Results are expressed as the mean number of osteoclasts per mm² or Msper mm² of diaphyseal intramedullary space, respectively.

Femora containing ACE-1 cells were imaged using bright field microscopyon a Nikon E600 fluorescence microscope equipped with a SPOT II digitalcamera utilizing SPOT image capture software (Diagnostic Instruments,Sterling Heights, Mich.). The total area of intramedullary space and thepercent of intramedullary space occupied by tumor, bone formation, andremaining hematopoeitic cells was calculated using Image Pro Plus v3.0software (Media Cybernetics, Silver Spring, Md.). Sabino et al., CancerRes. 62: 7343–7349, 2002; Sevcik et al., Pain 111: 169–180, 2004. Boneformation was analyzed using the same H&E stained femora sections usedto quantify tumor growth. Femur sections were viewed under polarizedlight to identify regions of woven and lamellar bone formation. Regionsof woven bone formation were imaged with the SPOTII digital camera andquantified using Image Pro Plus v3.0 software. Results are presented asarea of tumor, tumor-induced bone formation, and remaining hematopoeiticcells as a percentage of total intramedullary area.

Quantification of sensory fibers in bone and skin. The number of sensorynerve fibers was determined as previously described. Mach et al.,Neuroscience 113: 155–166, 2002. Briefly, the number of CGRP-IR fibersin three bone regions (proximal, distal and diaphyseal) and the threebone tissues (periosteum, mineralized bone and marrow) were identifiedusing a MRC-1024 Confocal Imaging system (Bio-Rad, Richmond, Calif.)equipped with a 20× objective. Nerve fiber counts were performed byviewing six femur sections per mouse with an Olympus BH-2fluorescence-equipped microscope. Only nerve fibers greater than 30 μmwere included in the analysis. To measure the total surface area (mm²)of each bone, we analyzed the same femur sections from which nerve fibercounts were obtained. The total bone area was measured on digital imagesof the femur sections acquired using a SPOTII digital camera and ImagePro Plus v.3.0 software. Results are presented as the number of fiberscounted per total bone area.

Quantification of epidermal innervation density was performed on 4randomly selected plantar hindpaw skin sections per mouse. The totalnumber of CGRP, TOH and RT97-IR nerve fibers were counted at 200×magnification. Counting rules were established to count only singleintra-epidermal fibers and not multiple branches of the same fiber.McCarthy et al., Neurology 45: 1848–1855, 1995. The total length ofepidermis in all sections quantified was measured using a 1 cm² eyepiecegrid. Only nerve fibers that were at least 30 μm in length, andprojected into the epidermis were counted. Results are given as the meannumber of intra-epidermal nerve fibers per mm length per mouse.

RC PCR analysis of mRNA levels of NGF in ACE-1 cells. Total RNA from dogbrain or dog prostate tumor cells ACE-1 was prepared according tomanufacturer's instructions using the RNeasy micro kit (Qiagen), and theRNA was quantified using Ribogreen reagent (Molecular Probes). Two-stepRT-PCR was performed using the TaqMan Gold RT-PCR kit (AppliedBiosystems). The RNA was reverse transcribed using random hexamers, andthe cDNA was amplified using a primer/probe set specific for NGF (LB041:AACAGGACTCACAGGAGCAA (SEQ ID NO:6), LB042: CGGCACTTGGTCTCAAAGAA (SEQ IDNO:7), and LB045: AATGTTCACCTCTCCCAGCACCATCA (SEQ ID NO:8)). The sampleswere analyzed in duplicate from the RT step and normalized to total RNAinput.

Statistical analysis. The Statview computer statistics package (SASInstitute, Inc., Cary, N.C.) was used to perform statistical tests.One-way ANOVA was used to compare behavioral results, bone histologicalresults, and immunohistochemical measures among the experimental groups.For multiple comparisons, Fishers's PLSD (protected least significantdifference) post hoc test was used. Significance level was set atP<0.05. The individual investigator responsible for behavior,immunohistochemistical analysis and scoring bone remodeling was blind tothe experimental situation of each animal.

Results

Anti-NGF therapy attenuated bone cancer pain to a greater extent thanmorphine sulfate but did not affect baseline thermal or mechanicalthresholds. Ongoing pain was analyzed by measuring spontaneous guardingand flinching over a 2-minute time period. ACE-1+vehicle micedemonstrated a greater time spent guarding (7.7±0.8 sec, day 19) ascompared to the sham+vehicle controls (0.6±0.3 sec, day 19, FIG. 10A).Additionally, ACE-1+vehicle mice exhibited an increased number offlinches (11.9±1.2, day 19) as compared to sham+vehicle controls(1.0±0.4, day 19, FIG. 10B). Administration of anti-NGF inACE-1-injected mice significantly attenuated spontaneous guarding(1.2±0.4 sec, day 19) as compared to ACE-1+vehicle mice (FIG. 10A).Anti-NGF treatment also significantly reduced spontaneous flinching inACE-1-injected mice (2.1±0.7, day 19) as compared to ACE-1+vehicle (FIG.10B). In preliminary studies, no significant behavioral differences orside effects were observed between sham-operated controls receivingeither vehicle or anti-NGF.

Anti-NGF therapy had no effect on either normal thermal response(10.2±0.4 sec, day 19) as compared to naïve+vehicle (11.2±0.4 sec, day19, FIG. 10C) or normal mechanical response (5.4±0.3 g, day 19) ascompared to naïve+vehicle (5.2±0.4 g, day 19, FIG. 10D).

Animals were tested to compare the efficacy of morphine sulfate (MS) tothe anti-NGF antibody in reducing bone cancer-related behaviors.Behavioral assessment on days 11 and 19 post-tumor injection revealedthat ACE-1+vehicle animals showed statistically longer time guarding ofthe injected limb (6.0±1.0 and 7.6±1.2 sec, day 11 and 19, respectively)compared to sham+vehicle mice (0.4±0.2 and 0.6±0.3 sec, day 11 and 19,respectively, FIG. 10E). ACE-1+vehicle also showed statistically largernumber of flinches of the injected limb (8.6±1.2 and 11.7±1.7, day 11and 19, respectively) compared to sham+vehicle mice (0.7±0.3 and1.0±0.4, day 11 and 19, respectively, FIG. 10F). Ongoing guarding wassignificantly reduced by either chronic treatment of anti-NGF (2.1±1.1and 1.4±0.4 sec, day 11 and 19, respectively), acute 10 mg/kg morphinesulfate (3.5±0.3 and 4.0±0.5 sec, day 11 and 19, respectively) or acute30 mg/kg morphine sulfate (2.2±0.3 and 2.0±0.4 sec, on day 11 and 19,respectively), as compared to ACE-1+vehicle mice (FIG. 10E). Ongoingflinching was also significantly reduced by either chronic treatment ofanti-NGF (3.4±1.7 and 2.6±0.6, day 11 and 19, respectively), acute 10mg/kg morphine sulfate (5.6±0.5 and 6.8±0.7, day 11 and 19,respectively) or acute 30 mg/kg morphine sulfate (3.6±0.5 and 3.5±0.7,day 11 and 19, respectively), as compared to ACE-1+vehicle mice (FIG.10F). Anti-NGF therapy significantly attenuated the bone cancer-relatedpain behaviors more effectively than acute 10 mg/kg morphine sulfate. Nodifferences in terminal weights were observed between sham+vehicle (27±1g), ACE-1+vehicle (27±1 g), and ACE-1+Anti-NGF (26±1 g) animals. Inthese studies, no significant behavioral differences or side effects,such as ataxia, illness, or lethargy, were observed between animalsreceiving either vehicle or anti-NGF.

Anti-NGF therapy attenuated touch-evoked bone cancer pain. Touch-evokedpain behavior was also assessed. Palpation-induced guarding andflinching were measured after the 2 min period of normally non-noxiouspalpation of the distal femur in ACE-1 and sham-injected animals. Asshown in FIGS. 10G and 10H, ACE-1-injected animals (administered withsaline) developed touch-evoked pain behaviors by day 7 as assessed bypalpation-induced guarding (FIG. 10G) and palpation-induced flinching(FIG. 10H) (both p<0.01, ANOVA) as compared to sham-injected animals(administered with saline). FIGS. 10G and 10H also shows that i.p.administration of anti-NGF antibody 911 significantly reducedpalpation-induced guarding (FIG. 10G) and palpation-induced flinching(FIG. 10H) in ACE-1-injected mice from day 11 to day 19 post-ACE-1 tumorimplantation as compared to administration of saline to ACE-1-injectedmice (p<0.01, ANOVA, for both palpation-induced guarding andpalpation-induced flinching). These results indicate anti-NGF antibody911 reduces touch-evoked pain in ACE-1-injected mice.

Anti-NGF therapy had no effect on markers of disease progression ortumor-induced bone formation. The effects of anti-NGF therapy on boneformation and destruction, tumor growth (FIG. 11), and osteoclastproliferation (FIG. 12) were examined 19 days post-tumor injection(Table 4 below). Sham-injected mice did not demonstrate significant boneremodeling (normalized transmission value of 115±2%) (FIG. 11A),osteoclast proliferation throughout the entire intramedullary space(16±10 osteoclasts/mm² diaphyseal intramedullary area) (FIG. 12A) ortumor cells (0±0%) (FIG. 11D), as assessed by radiological, TRAP and H&Eanalysis, respectively, as compared to ACE-1-injected mice. InACE-1+vehicle mice, there was extensive, but nearly equivalent boneformation and destruction as observed and characterized by multifocaldiaphyseal bridging and radiolucencies (normalized transmission value of109±5%) (FIG. 11B), marked increase in the number of osteoclasts (FIG.12B) and osteoblasts throughout the diaphyseal intramedullary area (47±3osteoclasts/mm² and 127±7 osteoblasts/mm²) and the tumor had filled mostof the intramedullary space (60±7% of intramedullary space) (FIG. 11E).Treatment of tumor-bearing mice with anti-NGF antibody from day 7 posttumor injection resulted in no significant change in bone remodeling(normalized transmission value of 106±9%) (FIG. 11C), no reduction inACE-1-induced osteoclast (FIG. 12C) or osteoblast proliferationthroughout the diaphyseal intramedullary area (47±5 osteoclasts/mm² and118±15 osteoblasts/mm²) or tumor growth (57±6% of intramedullary space)as compared to ACE-1+vehicle animals (FIG. 11F).

TABLE 4 Histological & Radiological quantification of bone remodelingand tumor progression in Anti-NGF and Vehicle treated ACE-1 AnimalsNaive + Sham + ACE-1 + ACE-1 + vehicle vehicle vehicle anti-NGF 1. BoneHistomorphometry Osteoclasts 7 ± 1 16 ± 10 47 ± 3^(a,b) 47 ± 5^(a,b) (OC#/mm² diaphyseal intramedullary space) Osteoblasts 81 ± 4  72 ± 5  127 ±7^(a,b)  118 ± 15^(a,b) (OB #/mm² diaphyseal intramedullary space)Macrophages (Ms) 2 ± 1 2 ± 1 27 ± 2^(a,b) 24 ± 3^(a,b) (Ms/mm²diaphyseal intramedullary space) Tumor-Induced New Bone Formation 0 ± 00 ± 0 14 ± 2^(a,b) 13 ± 1^(a,b) (% Diaphyseal Intramedullary spaceoccupied) Tumor Cells 0 ± 0 0 ± 0 60 ± 7^(a,b) 57 ± 6^(a,b) (%Intramedullary space occupied) Hematopoetic Cells 100 ± 0  100 ± 0  26 ±8^(a,b) 30 ± 6^(a,b) (% Intramedullary space occupied) 2. RadiologicalBone Remodeling Score % Normalized Transmission 100 ± 2  115 ± 2  109 ±5   106 ± 9   $\frac{\begin{matrix}\left( {1/\left( {{antilog}\;\left\lbrack {Optical} \right.} \right.} \right. \\\left. \left. {Density} \right\rbrack \right)\end{matrix}}{\begin{matrix}\left( {Naive} \right. \\\left. {Transmission} \right)\end{matrix}} \times 100\%$ ^(a)P < 0.05 versus naive. ^(b)P < 0.05versus sham (one way ANOVA, Fisher's PLSD).

Nineteen days following tumor injection, ACE-1+vehicle mice displayed anincrease in microphage (Ms) (27±2 Ms/mm² diaphyseal intramedullary area)as compared to sham+vehicle control mice (2±1 Ms/mm²). Anti-NGFtreatment of ACE-1-injected mice (24±3 Ms/mm²) did not significantlyalter Ms infiltration, as seen in the ACE-1+vehicle mice (Table 4).

Anti-NGF therapy has no observable effect on sensory or sympatheticinnervation in bone or skin. Thinly myelinated or unmyelinatedpeptidergic sensory nerve fibers (CGRP-IR), large myelinated sensoryfibers (RT97-IR) and noradrenergic sympathetic nerve fibers (TOH-IR)were analyzed in the ACE-1 injected femora or the hindpaw plantar skinby immunohistochemistry using antibodies raised against CGRP, RT-97 andTOH, respectively. CGRP-IR nerve fibers were found throughout the entirebone (periosteum, mineralized bone. bone marrow and tumor) of theACE-1+vehicle (23.5±1.9 fibers/mm²) and ACE-1+anti-NGF (24.0±1.9fibers/mm²) animals as well as in the sham+vehicle (28.2±1.5 fibers/mm²)and naïve+vehicle animals (24.6±2.4 fibers/mm²) or naïve+anti-NGFanimals (23.1±1.9 fibers/mm²) (FIG. 14). There was no significantdifference between the intensity or density of CGRP-IR fibers inACE-1+vehicle (13.9±0.5 fibers/mm) and ACE-1+anti-NGF (15.2±0.7fibers/mm) hindpaw skin samples (FIGS. 13A&B). Similarly, there was nosignificant difference between the intensity or density of CGRP-IRfibers in naïve+vehicle (14.4±0.4 fibers/mm) and naïve+anti-NGF(14.2±1.3 fibers/mm) hindpaw skin samples (FIGS. 14A&B). Differences inthe density and intensity of RT97-IR and TOH-IR fibers were alsoundetectable in naïve+vehicle (4.2±2.2 RT97+fibers/mm; 16.0±2.7 TOH+fibers/mm) and naïve+anti-NGF treated (8.0±0.6 RT97+ fibers/mm; 12.8±1.1TOH+ fibers/mm) animals. There were no significant observabledifferences between the intensity or density of CGRP, RT97 or TOH-IRfibers in the skin samples of ACE-1+vehicle and ACE-1+anti-NGF versusthe naïve+vehicle and naïve+anti-NGF animals.

Level of mRNA expression in ACE-1 cells. NGF expression in dog brain andACE-1 cells were compared. Five independent ACE-1 samples were analyzed,and in each one NGF expression was below the level of detection of thePCR assay. NGF in dog brain crossed the threshold at cycle 35.2 of a 40cycle experiment, whereas the ACE-1 samples failed to cross thethreshold after 40 cycles. Thus, NGF mRNA expression in the ACE-1samples was at least 27.8 fold less than expression in brain.

Example 4 Analgesic Effects of Anti-NGF Antibody E3 in Patients withModerate to Severe Pain from Metastases to the Bone Due to EitherProstate or Breast Cancer

In a randomized, placebo-controlled, double-blind study, analgesiceffects (including time to onset, time to peak, duration as well as painrelief as measured by Visual Analogue Scale (VAS)) of intravenous doses(100 μg/kg, 300 μg/kg, or 1,000 μg/kg) of anti-NGF antibody E3 arecompared with placebo in patients with moderate to severe pain frommetastases to the bone due to either prostate or breast cancer. Adultmales and females (ages between 35 to 75) who are experiencingmoderate-to-severe pain from metastases to the bone due to eitherprostate or breast cancer, are enrolled in the study. During thescreening period, patients are required to record their pain level fourtimes per day and also record their use of other analgesic medicationfor 14 days prior to administration of anti-NGF antibody E3.

Two hundred and eighty patients are admitted to the study. Anti-NGFantibody E3 administration occurs on the mornings of Day 1 and Day 29,following the recording of two weeks of baseline pain level, otheranalgesic use, and adverse events. Two hundred and eighty patients aredivided into four groups and each group has seventy patients. Patientsin each group are treated with placebo, 100 μg/kg, 300 μg/kg, or 1,000μg/kg of anti-NGF antibody E3.

Analgesic effects are assessed four times daily for fourteen days beforedosing and for a period of six months after administration of antibodyE3. Outcome is assessed as change from screening baseline (mean painlevel for fourteen days prior to administration of placebo or antibodyE3). Any decrease in pain scores and/or decrease in use of otheranalgesics in one or more groups of patients treated with anti-NGFantibody E3 compared to placebo demonstrate efficacy of the treatmentwith anti-NGF antibody E3.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention.

1. A method for treating bone cancer pain in an individual comprisingadministering to the individual an effective amount of a nerve growthfactor (NGF) antagonist.
 2. The method of claim 1, wherein the bonecancer pain is from cancer originated in bone.
 3. The method of claim 2,wherein the bone cancer pain is from osteosarcoma.
 4. The method ofclaim 1, wherein the bone cancer pain is from cancer metastasized tobone.
 5. The method of claim 4, wherein the bone cancer pain is fromprostate cancer metastasized to bone.
 6. The method of claim 4, whereinthe bone cancer pain is from breast cancer metastasized to bone.
 7. Themethod of claim 4, wherein the bone cancer pain is from lung cancermetastasized to bone.
 8. The method of claim 4, wherein the bone cancerpain is from sarcoma metastasized to bone.
 9. The method of claim 4,wherein the bone cancer pain is from renal cancer metastasized to bone.10. The method of claim 1, wherein the NGF antagonist is an anti-NGFantagonist antibody.
 11. The method of claim 10, wherein the anti-NGFantagonist antibody is a monoclonal antibody.
 12. The method of claim10, wherein the anti-NGF antagonist antibody is a humanized antibody.13. The method of claim 10, wherein the anti-NGF antagonist antibody isa human antibody.
 14. The method of claim 10, wherein the anti-NGFantagonist antibody binds human NGF.
 15. The method of claim 14, whereinthe anti-NGF antagonist antibody further binds rodent NGF.
 16. Themethod of claim 14, wherein the anti-NGF antagonist antibody binds humanNGF with a K_(D) of about 0.1 nM or less than about 0.1 nM.
 17. Themethod of claim 1, wherein the NGF-antagonist is not co-administeredwith an opioid analgesic.